Laser Capture in Microscopy and Microdissection,

Contents Contributors to Volume 356 • MISCELLANEOUS Pages ix-xii Preface • EDITORIAL Page xiii P. Michael Conn Volumes i...

Author: P. Michael Conn

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Contents Contributors to Volume 356 • MISCELLANEOUS Pages ix-xii Preface • EDITORIAL Page xiii P. Michael Conn Volumes in series • MISCELLANEOUS Pages xv-xxxv [1] Comparison of current equipment • ARTICLE Pages 3-12 Anda Cornea and Alison Mungenast [2] Laser capture microdissection and its applications in genomics and proteomics • ARTICLE Pages 12-25 James L. Wittliff and Mark G. Erlander [3] Going in vivo with laser microdissection • ARTICLE Pages 25-33 Anette Mayer , Monika Stich , Dieter Brocksch , Karin Schütze and Georgia Lahr [4] Use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis • ARTICLE Pages 33-49 Rachel A. Craven and Rosamonde E. Banks [5] Optimized tissue processing and staining for laser capture microdissection and nucleic acid retrieval • ARTICLE Pages 49-62 Lora E. Huang , Veronica Luzzi , Torsten Ehrig , Victoria Holtschlag and Mark A. Watson [6] Fluorescence in Situ hybridization of LCM-isolated nuclei from paraffin sections • ARTICLE Pages 63-69 Douglas J. Demetrick , Sabita K. Murthy and Lisa M. DiFrancesco [7] Immunoblotting of single cell types isolated from frozen sections by laser microdissection • ARTICLE Pages 70-79 Livia Casciola-Rosen and Kanneboyina Nagaraju

[8] Noncontact laser Catapulting: A basic procedure for functional genomics and proteomics • ARTICLE Pages 80-99 Gabriela Westphal , Renate Burgemeister , Gabriele Friedemann , Axel Wellmann , Nicolas Wernert , Volker Wollscheid , Bernd Becker , Thomas Vogt , Ruth Knüchel , Wilhelm Stolz and Karin Schütze [9] Internal standards for laser microdissection • ARTICLE Pages 99-113 Ludger Fink and Rainer Maria Bohle [10] Methacarn: A fixation tool for multipurpose genetic analysis from paraffin-embedded tissues • ARTICLE Pages 114-125 Makoto Shibutani and Chikako Uneyama [11] Use of laser capture microdissection for clonal analysis • ARTICLE Pages 129-136 Valerie Paradis and Pierre Bedossa [12] Laser capture microdissection in carcinoma analysis • ARTICLE Pages 137-144 Yen-Li Lo and Chen-Yang Shen [13] Laser capture microdissection to assess development • ARTICLE Pages 145-156 Carlos A. Suárez-Quian , Oscar M. Tirado , Francina Munell and Jaume Reventós [14] Application of laser capture microdissection to proteomics • ARTICLE Pages 157-167 K. K. Jain [15] Laser capture microdissection of mouse intestine: Characterizing mrna and protein expression, and profiling intermediary metabolism in specified cell populations • ARTICLE Pages 167-196 Thaddeus S. Stappenbeck , Lora V. Hooper , Jill K. Manchester , Melissa H. Wong and Jeffrey I. Gordon [16] Laser capture microdissection in pathology • ARTICLE Pages 196-206 Falko Fend , Katja Specht , Marcus Kremer and Leticia Quintanilla-Martínez [17] Use of laser capture microscopy in the analysis of mouse models of

human diseases • ARTICLE Pages 207-215 Meral J. Arin and Dennis R. Roop [18] Use of laser microdissection in complex tissue • ARTICLE Pages 216-223 Holler S. Willenberg , Rhodri Walters and Stefan R. Bornstein [19] Assessment of clonal relationships in malignant lymphomas • ARTICLE Pages 224-240 Kojo S. J. Elenitoba-Johnson [20] Comparison of normal and tumor cells by laser capture microdissection • ARTICLE Pages 240-247 Jaume Mora , Muzaffar Akram and William L. Gerald [21] Analysis of folliculostellate cells by laser capture microdissection and reverse transcription-polymerase chain reaction (LCM-RT/PCR) • ARTICLE Pages 248-255 Ricardo V. Lloyd , Long Jin , Katharina H. Ruebel and Jill M. Bayliss [22] Analysis of gene expression • ARTICLE Pages 259-270 Janette K. Burgess and Brent E. McParland [23] Analysis of specific gene expression • ARTICLE Pages 271-281 Georgia Lahr , Anna Starzinski-Powitz and Anette Mayer [24] Gene Discovery with laser Capture Microscopy • ARTICLE Pages 282-289 Mauricio Neira and Edwin Azen [25] DNA fingerprinting from cells captured by laser microdissection • ARTICLE Pages 289-294 Yongyut Sirivatanauksorn , Vorapan Sirivatanauksorn and Nicholas R. Lemoine [26] Single cell PCR in laser capture microscopy • ARTICLE Pages 295-301 Sinuhe Hahn , Xiao Yan Zhong and Wolfgang Holzgreve

[27] Assessment of genetic heterogeneity in tumors using laser capture microdissection • ARTICLE Pages 302-309 Dave S. B. Hoon , Akihide Fujimoto , Sherry Shu and Bret Taback [28] Gene mutations: Analysis in proliferative prostatic diseases using laser capture microdissection • ARTICLE Pages 309-322 Hitoshi Takayama , Norio Nonomura and Katsuyuki Aozasa [29] Use of laser capture microdissection-generated targets for hybridization of high-density oligonucleotide arrays • ARTICLE Pages 323-333 Hiroe Ohyama , Mamatha Mahadevappa , Heikki Luukkaa , Randy Todd , Janet A. Warrington and David T. W. Wong [30] Single cell gene mutation analysis using laser-assisted microdissection of tissue sections • ARTICLE Pages 334-343 Åsa Persson , Helena Backvall , Fredrik Pontén , Mathias Uhlén and Joakim Lundeberg [31] methylation in gene promoters: Assessment after laser capture microdissection • ARTICLE Pages 343-351 Arthur R. Brothman and Jiang Cui Author Index • MISCELLANEOUS Pages 353-373 Subject Index • MISCELLANEOUS Pages 375-385

Contributors to Volume 3 5 6 Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

MUZAFFAR AKRAM (20), Department of

ARTHUR R. BROTHMAN (31), Departments

Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 KATSUYUKI AOZASA (28), Department of Pathology, Osaka University Medical School Suita, Osaka 565-0871, Japan MERAL J. ARIN (17), Department of Dermatology, University of Cologne, 50924 Cologne, Germany EDWIN AZEN (24), Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706 HELENA B~,CKVALL (30), Department of Genetics and Pathology, University Hospital S-751 85 Uppsala, Sweden ROSAMONDE E. BANKS (4), Cancer Research UK Clinical Centre, St. James University Hospital Leeds LS9 7TE United Kingdom JILL M. BAYLISS (21), Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 BERND BECKER (8), Department of Dermatology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany PIERRE BEDOSSA (11), Faculty of Pharmacy, CNRS ESA 8067, 75005 Paris, France RAINER MARIA BOHLE (9), Institute of Pathology, Justus-Liebig-Universitiit Giessen, 35392 Giessen, Germany STEFAN R. BORNSTEIN(18), Department of Endocrinology, University of Diisseldorf, D-40225 Diisseldorf, Germany DIETER BROCKSCH(3), Servicebereich Corporate Communications, Carl Zeiss, D73447 Oberkochen, Germany

of Pediatrics and Human Genetics, University of Utah Health Sciences Center, Salt Lake City, Utah 84132 RENATE BURGEMEISTER(8), PA.L.M. Mi-

crolaser Technologies AG, 82347 Bernried, Germany JANETrE K. BURGESS (22), Respiratory Re-

search Group, Department of Pharmacology, University of Sydney, Sydney, New South Wales, 2006 Australia LIVlA CASCIOLA-ROSEN(7), Department of

Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 ANDA CORNEA (1), Oregon National Pri-

mate Research Center, Beaverton, Oregon 97006 RACHEL A. CRAVEN (4), Cancer Research

UK Clinical Centre, St. James University Hospital Leeds LS9 7TF, United Kingdom JIANG CUI (31), Departments of Pediatrics

and Human Genetics, University of Utah Health Sciences Center, Salt Lake City, Utah 84132 DOUGLAS J. DEMETRICK(6), Calgary Lab-

oratory Services, Departments of Pathology, Oncology, and Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada LISA M. DIFRANCESCO (6), Calgary Labo-

ratory Services, Departments of Pathology, Oncology, and Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada ix

X

CONTRIBUTORS TO VOLUME 356

TORSTENEHRIG (5), Department of Derma-

LORA V. HOOPER (15), Department of

tology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

KOJO S. J. ELENITOBA-JOHNSON(19), Di-

vision of Anatomic Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132

LORA E. HUANG(5), Department of Internal

MARK G. ERLANDER (2), Arcturus Ap-

K. K. JAIN (14), Jain PharmaBiotech, CH-

plied Genomics, West Carlsbad, California 92008 FALKO FEND (16), Institute of Pathol-

ogy, Technical University Munich, D-81675 Munich, Germany LUDGER FrNK (9), Institute of Pathol-

Medicine, University of lowa, Iowa City, Iowa 52246 4057 Basel, Switzerland LONG JIN (21), Department of Laboratory

Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 RUTH KNOCHEL (8), Department of Der-

ogy, Justus-Liebig-Universitiit Giessen, 35392 Giessen, Germany

matology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany

GABRIELE FRIEDEMANN (8), P.A.L.M. Mi-

MARCUS KREMER (16), Institute of Pa-

erolaser Technologies AG, 82347 Bernried, Germany AKIHIDE F~J1MOTO (27), Department of

Molecular Oncology, John WayneCancer Institute, Santa Monica, California 90404 WILLIAM L. GERALD (20), Department

of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 JEFFREY I. GORDON (15), Department of

Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 SINUHE HAHN (26), Laboratory for Pre-

natal Medicine, Department of Obstetrics and Gynecology, University of Basel, CH-4031 Basel, Switzerland VICTORIA HOLTSCHLAG (5), The Siteman

Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, Missouri 63110 WOLFGANG HOLZGREVE (26), Laboratory

for Prenatal Medicine, Department of Obstetrics and Gynecology, University of Basel, CH-4031 Basel, Switzerland DAVE S. B. HOON (27), Department of

Molecular Oncology, John WayneCancer Institute, Santa Monica, California 90404

thology, Technical University Munich, D-81675 Munich, Germany GEORGIA LAHR (3, 23), Laser Labora-

tory and Department of Molecular Biology, Staedtisches KrankenhausMiinchenHarlaching, D-81545 Munich, Germany NICHOLAS R. LEMOINE (25), Cancer Re-

search UKMolecular Oncology Unit, Department of Cancer Medicine, Imperial College of Science, Technology, and Medicine, London W12 ONN, United Kingdom RICARDO V. LLOYD (21), Department

of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 YEN-LI LO (12), Institute of Biomedical

Sciences, Academia Sinica, Taipei 11529, Taiwan Department of Biotechnology, Royal Institute of Technology (KTH), SCFAB, S-106 91 Stockholm, Sweden

JOAKIM LUNDEBERG (30),

HEIKKI LUUKKAA (29), Division of Oral

Pathology, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, Massachusetts 02115


xi

VERONICALUZZI (5), Department of Pathol-

VALt~RIEPARADIS (11), Department of Pa-

ogy and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 MAMATHA MAHADEVAPPA(29), Affymetrix Inc., Santa Clara, California 95051 JILL K. MANCHESTER (15), Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 ANETrE MAYER (3, 23), Humangenetik fiir Biologen, Universitiit Frankfurt, D60054 Frankfurt~Main, Germany BRENT E. MCPARLAND (22), Department of Pathology, University of Sydney, Sydney, New South Wales, 2006Australia JAUME MORA (20), Department of Hematology and Oncology, Hospital Sant Joan de Deu de Barcelona, Barcelona, Spain FRANCINA MUNELL (13), Unitat de Recerca Biomddica, Hospital MaternoInfantiL Vall d'Hebron Hospital 08035 Barcelona, Spain ALISON MUNGENAST (1), Oregon Health and Science University, Portland, Oregon 97201, and Oregon National Primate Research Center, Beaverton, Oregon 97006 SABITA K. MURTHY (6), Departments of Pathology, Oncology, and Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada KANNEBOY1NA NAGARAJU (7), Division of Rheumatology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 MAURICIO NEIRA (24), Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada NORIO NONOMURA (28), Department of Urology, Osaka University Medical School Suita, Osaka 565-0871, Japan HIROE OHYAMA (29), Division of Oral Pathology, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, Massachusetts 02115

thology, Beaujon Hospital 92110 Clichy, France ASA PERSSON (30), Department of Biotech-

nology, Royal Institute of Technology (KTH), SCFAB, S-106 91 Stockholm, Sweden FREDRIK PONTI~N (30), Department of

Genetics and Pathology, University Hospital S-751 85 Uppsala, Sweden LETICIA QUINTANILLA-MARTfNEZ(16), De-

partment of Pathology, GSF Research Center for Environment and Health, D-85758 Oberschleissheim, Germany JAUME REVENT6S (13), Unitat de Re-

cerca Biomkdica, Hospital MaternoInfantiL Vail d'Hebron Hospital 08035 Barcelona, Spain DENNIS R. ROOP (17), Departments of

Molecular and Cellular Biology and Dermatology, Baylor College of Medicine, Houston, Texas 77030 KATHARINA H. RUEBEL (21), Department

of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 KARIN SCHgrrZE (3, 8), PA.L.M. Microlaser

Technologies AG, 82347 Bernried, Germany CHEN-YANG SHEN (12), Institute of Biomed-

ical Sciences, Academia Sinica, Taipei 11529, Taiwan MAKOTO SHIBUTANI (10), Division of

Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan SHERRY SHU (27), Department of Molecular

Oncology, John Wayne Cancer Institute, Santa Monica, California 90404 VORAPAN SIRIVATANAUKSORN(25), Faculty of Medicine, Mahidol University, Bangkok 10700, Thailand YONGYUT SIRIVATANAUKSORN(25), Fac-

ulty of Medicine, Mahidol University, Bangkok 10700, Thailand

xii


KATJA SPECHT (16), Institute of Pathology,

Technical University Munich, D-81675 Munich, Germany THADDEUS S. STAPPENBECK(15), Depart-

ment of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 ANNA STARZINSKI-POWITZ(23), Human-

genetik fiir Biologen, Universiti~t Frankfurt, D-60054 Frankfurt~Main, Germany MONIKA STICH (3), Laser Laboratory

and Department of Molecular Biology, Staedtisches Krankenhaus MiinchenHarlachin g, D-81545 Munich, Germany WILHELM STOLZ (8), Department of Der-

matology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany CARLOS A. SU.~REZ-QUIAN(13), Depart-

ment of Cell Biology, Georgetown University Medical School Washington, D. C. 20007 BRET TABACK(27), Department of Molecu-

lar Oncology, John Wayne Cancer Institute, Santa Monica, California 90404 HITOSHI TAKAYAMA(28), Department of

Pathology, Osaka University Medical School, Suita, Osaka 565-0871, Japan OSCAR M. TIRADO (13), Unitat de Re-

cerca BiomOdica, Hospital MaternoInfantil, Vail d'Hebron Hospital 08035 Barcelona, Spain RANDY TODD (29), Division of Oral Pathol-

ogy, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, Massachusetts 02115 MATHIAS UHLt~N (30), Department of

Biotechnology, Royal Institute of Technology (KTH), SCAFB, S-106 91 Stockholm, Sweden CHIKAKO UNEYAMA (10), Division of

Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan

THOMAS VOGT (8), Department of Der-

matology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany RHODRI WALTERS (18), Department of Endocrinology, University of Diisseldorf, D40225 Diisseldorf, Germany JANET m. WARRINGTON (29), Affymetrix Inc., Santa Clara, California 95051 MARK A. WATSON (5), Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 AXEL WELLMANN (8), Institute of Pathology, University of Bonn, 53011 Bonn, Germany NICOLAS WERNERT(8), Institute of Pathology, University of Bonn, 53011 Bonn, Germany GABRIELA WESTPHAL(8), P.A.L.M. Microlaser Technologies AG, 82347 Bernried, Germany HOLGER S. WILLENBERG (18), Department of Endocrinology, University of Diisseldorf, D-40225 Diisseldorf, Germany JAMES L. WITrL1FF (2), Hormone Receptor Laboratory, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202 VOLKER WOLLSCHEID (8), Ciphergen Biosystems Ltd., Surrey Technology Centre, Guildford, Surrey GU2 7YG, United Kingdom DAVID T. W. WONG (29), University of California School of Dentistry, Dental Research Institute, LOs Angeles, California 90095 MELISSA H. WONG (15), Department of Dermatology, Cell and Development Biology, Oregon Health and Science University, Portland, Oregon 97201 XIAO YAN ZHONG (26), Laboratory for Prenatal Medicine, Department of Obstetrics and Gynecology, University of Basel CH-4031 Basel Switzerland

Preface

Five years ago few people had heard of"laser microdissection" or "laser capture microscopy." Now most major institutions have it as a core facility. This volume documents many diverse uses for this technique in disciplines that broadly span biology. The methods presented include shortcuts and conveniences not included in the sources from which they were taken. To the degree possible, we have included information needed to select equipment, prepare samples, and analyze data. The techniques are described in a context that allows comparisons to other related methodologies. The authors were encouraged to do this in the belief that such comparisons are valuable to readers who must adapt extant procedures to new systems. Also, so far as possible, methodologies are presented in a manner that stresses their general applicability and potential limitations. Although for various reasons some topics are not covered, the volume provides a substantial and current overview of the extant methodology in the field and a view of its rapid development. Particular thanks go to the authors for their attention to meeting deadlines and for maintaining high standards of quality, to the series editors for their encouragement, and to the staff of Academic Press for their help and timely publication of this volume. P. MICHAEL CONN

xiii

[11

COMPARISON OF CURRENT EQUIPMENT

3

[1] Comparison of Current Equipment By ANDA

CORNEA a n d ALISON MUNGENAST

Introduction Some of the most exciting new developments in biomedical research, such as DNA microarrays and proteomics, depend on the isolation of single cells or pure populations of cells with specific phenotypes. Several microscopic techniques are currently used for microdissection: suction of a cell content through micron-size glass pipettes, dissection using a piezo-activated metal knife and suction through a glass pipette (PPMD by Brinkmann), and dissection using lasers. Because of the high energy concentrated into a small area, the easy control of the beam position, and the lack of direct contact with the material to be dissected, lasers provide the best option for easy-to-use, large-scale microdissections. There are currently three commercially available systems designed specifically for laser capture microdissection: PixCell by Arcturus (Mountain View, CA), PALM by P.A.L.M. Mikrolaser Technologie (Wolfratshousen, Germany), and the Leica AS LMD by Leica (Heidelberg, Germany). The PixCell originated in a Cooperative Research and Development Agreement between the National Institutes of Health, the National Cancer Institute, and the National Institute for Child and Human Development and is now manufactured and marketed by Arcturus. The system uses an IR laser focused through a microscope objective to heat a plastic film placed above a section of tissue. The plastic melts temporarily in the small area irradiated and penetrates the tissue. When the laser beam is turned off, the plastic solidifies and forms bonds with the tissue it has penetrated. When the plastic sheet is removed, the tissue bonded to the plastic is removed as well and thereby isolated or dissected from the rest (Fig. I). The dissected material may then be processed for the isolation of RNA, DNA, or proteins (Emmert-Buck et al. I). The PALM system uses an N2 laser with 336 nm wavelength. The laser, also focused through an inverted microscope objective, has enough energy to ablate tissue or cells that are in focus. Ablation, which destroys chemical bonds within a tissue by a mechanism not fully understood, may remove undesirable cells or groups of cells and isolate a region of interest. When the laser is slightly defocused, its energy may be used to catapult the dissected material up to where it may be collected into a cap and stored or immediately used for the isolation of RNA, DNA, 1 M. R. Emmert-Buck, R. E Bonner, P. D. Smith, R. E Chuaqui, Z. Zuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996).

METHODSINENZYMOLOGY,VOL.356

Copyright2002,ElsevierScience(USA). Allrightsreserved. 0076-6879/02 $35.00

4

BASIC PRINCIPLES

[iI

Plastic film

Tissue section Glass slide

A

•

Dissected cell

B

Plastic film

Tissue section Glass slide

FIG. 1. Principle of laser capture microscopy used by Arcturus. (A) A thin plastic film is lowered over the specimen to be dissected. An IR laser beam focus through a microscope objectiveilluminates a small area on the plastic causing it to melt locally and penetrate the tissue. (B) When the laser is interrupted,the plastic solidifiesforming bonds with the underlyingtissue. When the plastic is removed, the attached tissue is isolated from the rest of the specimen.

or proteins (Schutze and Lahr2). This process, termed laser pressure catapulting, is patented by P.A.L.M. (Fig. 2). The Leica AS LMD, more recently introduced as the third generation of laser microdissection systems, uses a pulsed UV laser similar to the P A L M on an upright microscope. The laser beam may be moved with a software-controlled mirror system to select cells to be ablated or to isolate the area to be dissected. The dissected material is allowed to fall by gravity into a cap and may thereafter be used for isolating proteins or genetic material (Fig. 3). There are several parameters that may be used to characterize a laser microdissection system. A m o n g them the most important are the resolution and the specificity of dissection and the integrity of the dissected material. Very important also are the ease of use, the reliability of the instrument, and the quality of service and support. Resolution The smallest area that can be isolated from the rest o f a tissue by laser microdissection is related to the size of the laser beam and therefore depends on the numerical aperture (NA) of the objective used for dissection and on the wavelength of light used. The N A of an objective is defined as the sine o f the collection 2 K. Schute and G. Lahr, Nat. Biotechnol. 16, 737 (1998).

[ 1]

COMPARISONOF CURRENT EQUIPMENT

A

i

5

Tissue section Plastic film Glass slide

i T Laserbeam (ablating)

B

C

FIG. 2. Principleof the PALMlaser microdissection. (A) A UV laser beam focusedby the objective of an inverted microscopecuts a contour around the area to be dissected. (B) The laser is defocused and positioned within the selected area. (C) The laser pressure is used to lift the dissected sample into a collecting cap. This process, named laser pressure catapulting, is patented by P.A.L.M. angle multiplied by the refractive index of the immersion medium. Most common configurations for laser microdissection use dry objectives, which somewhat limits the NA. The most stringent limitation comes, however, from the maximum collection angle that can be used. For limited lens diameters, a large angle imposes a short working distance. In most cases, specimens to be dissected are mounted on standard microscope glass slides with approximately 1 m m thickness. For the PALM and the Leica AS LMD systems, the light exiting the objective needs to cross this distance in order to be focused on the tissue. This requires long working distance objectives that necessarily have a lower NA and consequently a wider laser beam waist. For cases when the thinnest possible cuts are required, higher NA objectives may be used if the specimen is mounted on a thin glass coverslip, making possible the use of short working distance objectives with high NA. The main inconvenience in this case is the fragility of these coverslips, as all steps prior to dissection, including collection of sections, fixing, staining, transport, and mounting on the microscope stage, have to be done with extra care. In the PixCell, light leaving the objective passes through the collecting cap and that also limits the minimum usable working distance and consequently the NA of

6

[ ]]

BASIC PRINCIPLES

Glass slide Plastic film Tissue section

~,

B

Laser beam (ablating)

I

C Dissected cell FIG. 3. Principle of the Leica AS LMD microdissection. (A) A laser beam similar to the one used by PALM is concentrated by the objective of an upright microscope. The specimen, mounted on a PEN membrane, is mounted upside down. (B) A contour is cut through the membrane and tissue around the area to be dissected by moving the laser beam and not the stage. (C) The dissected area, isolated from the rest of the specimen, falls into a collecting cap positioned under the specimen.

the objective. In this case, the user does not have the option to mount specimens on coverslips; the only improvement could come from redesigning the collection caps. The size of the beam waist depends on the wavelength of light used. The infrared light used by PixCell will give a larger beam waist than the UV light used by the PALM and Leica AS LMD for the same objective used. The demands for the smallest possible beam waist, however, are different for the two classes of microdissection instruments, as the dissection technique is different. For the PixCell, the beam waist gives the minimum dissected area. The minimum value cited by the manufacturer is 7.5/zm. This is about the size of a cell, or smaller than many cells. It can be argued that a smaller size is hardly necessary, as in most cases laser microdissection is used to isolate single cells or larger numbers of identical cells and, in this case, the intent is to collect as much material per cell as possible. The PALM and Leica AS LMD use the laser to ablate a contour line around a single cell and therefore the width of the cut is expected to be much smaller. The minimum cut sizes quoted for the PALM and Leica AS LMD were less than 1/zm and 2.5/zm, respectively.

[ 1]

COMPARISONOF CURRENTEQUIPMENT

7

Specificity and Integrity In all systems the material removed may include more than the specific area marked for dissection. In the case of the PixCeU, the bond between the plastic and the tissue must be stronger than the bond between tissue and slide in order to allow removal of the cell or tissue part marked for dissection. Uncharged and unsubbed slides work well because the tissue adheres more loosely to the slides. This presents a problem, however, when the user desires to process the tissue beyond simple staining, such as using immunohistochemistry, as sections may be lost during the procedure. The bonds within the tissue, dependent on tissue type and fixation, may also overcome in some cases the attachment to the slide, and extra material will be removed contaminating the purity of the dissected sample (Fig. 4). For some of the collecting caps, the most frequently used ones, the plastic film touches the section of tissue to be dissected and may remove material that randomly adhered to it. This is, however, overcome in the more newly designed "CapSure" caps in which only the melted film touches the tissue. Sticky "prep strips" are also provided which can reduce contamination.

FIG. 4. Positive selection of cells to be dissected with PixCell laser capture microscope. (A) Brain section in which cells selected for dissection appear in a lighter color after the plastic transfer film was attached by melting. (B) Cells removed with the film. Arrows indicate cells for which nuclei failed to transfer. Arrowheads indicate dissection that removed extra material. Note the clarity of quality control.

8

BASIC PRINCIPLES

[1]

mm FIG.5. Negative selection of area to be selected by a PALM laser microdissection microscope. (A) A freehand line is drawn on the screen over the image. (B) The laser cuts the PEN membrane and ablates the tissue along that line. (C) The tissue enclosedby the cut is catapulted using laser pressure. (D) Sample remaining after positive selection of single cells in a different tissue section mounted directly on a glass slide.

The laser ablation used in the PALM and Leica LDM is aimed at circumventing this contamination problem by destroying the tissue around the cell or the region of interest that can then be collected free of neighboring contamination (Fig. 5). The process of ablation itself is not well understood and the destruction of chemical bonds may not be complete. Particles of the material targeted for ablation may be seen, while cutting, landing on the cell or region to be dissected suggesting a potential for contamination. Cells adjacent to the cell of interest cannot be collected as well with this process, as they are ablated by the laser beam. With the Arcturus, however, it is possible to collect material from adjacent cells. The integrity of the material dissected may be an issue as in all cases the sample is irradiated with laser light that has the potential to alter chemical bonds by either destroying them or causing cross-linking. This is particularly an issue for the UV lasers even though the wavelength is slightly larger than the main absorption peak for proteins and nucleic acids. The high temperature created by the IR laser in the PixCell system, necessary for the melting of the film, may also degrade the dissected material. There is at this time a large body of literature suggesting that adequate RNA may be quantitatively isolated by both technologies and not much published evidence to the contrary.

Ease of Use There are many steps involved in laser capture microdissection that may affect the ease of use. The type of slides required, demands on sectioning and processing of the tissue, visualization and selection of the region to be selected, and dissection

[1]

COMPARISON OF CURRENTEQUIPMENT

9

and collection of the dissected material are all variables that may make the technique easy and straightforward or complicated and awkward. Regular microscope slides may be used in all systems, albeit with suboptimal results. For the PALM and the Leica DML it is recommended that a thin PEN (polyethylene naphthalate) membrane be mounted between the slide and the tissue. The membrane may be cut by the ablation laser around the area targeted for dissection and then catapulted in the PALM or let drop in the Leica, preserving the integrity of the cells attached to it. Dissected pieces of tissue may be visualized after capture in the collecting cap only if the underlying membrane kept the structure intact. If cells are catapulted directly from a slide, the material will be pulverized and it will be impossible to assess the efficiency of capture by visualization. PEN coated slides may be purchased but are relatively expensive. Preparing them, however, may be very time consuming. We have also found that the membranes tear easily during immunohistochemical processing. For the PixCell, the tissue may be mounted directly on a glass slide. The dissected cells are then collected on a plastic film attached to the collecting caps and can be easily visualized. For all systems, the efficiency of dissection and collection is critically dependent on tissue preparation as well as on environmental factors.

Sample Preparation Arcturus provides a number of protocols for sample preparation that must be strictly followed in order to obtain a good dissection. The thickness of the section, method of fixation of the tissue, and staining are restricted. Specimens need to be perfectly dry to adhere to the transfer film. The PALM and the Leica systems rely largely on the ablation of the PEN foil for dissection and therefore offer more freedom for the preparation of the specimen. Tissue sections may be much thicker and various fixations may be used. The PALM may even isolate live cells. Not everything works, however, and the greater freedom in sample preparation comes with the necessity for users to optimize their own protocols.

Environment Humidity in the specimen, due either to incomplete drying or to absorption of air vapors, makes dissection in the PixCell impossible. Samples and instrument need to be kept in a dry environment. Humidity between the slide and the membrane prevents proper membrane cutting in the PALM. Lack of humidity, however, contributes to increased electrostatic forces that compete with gravity and affect collection in the Leica AS LMD system. In this case, the instrument should be kept in a high-humidity room free of air drafts.

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[ 1]

Control of Stage Movement The control of the stage and selection of areas for dissection may strongly affect the ease of use. The PixCell uses a joystick manual control of the stage movement. The stage is moved so that each intended target is brought into the middle of the field of view, under the laser beam, and dissected, before moving to the next target and so on. If areas larger than can be covered by a single beam are desired, multiple laser shots need to be fired as the stage is moved. This requires skill and concentration, as any misfire can contaminate the whole sample. The PALM uses computer-controlled stage motors that can move the stage along a predrawn path. This is particularly convenient when large areas of irregular shapes are dissected. A freehand drawing tool allows the operator to outline the target area, and then the stage moves as the laser fires and cuts the underlying membrane along the chosen path. The whole area is then catapulted into the collecting cap. When several isolated cells are collected into the same cap, they can each be outlined first, then the system isolates them one by one, automatically. The Leica AS LMD uses a computer-controlled mirror that moves the laser beam along a path also preselected by a freehand drawing tool. Cutting may be in this case much faster than for the PALM, as the stage is immobile and the laser beam is moved faster.

Service A major factor in the satisfaction with any of the instruments chosen is the reliability and speed and quality of service. Arcturus has the longest history of operation and, at least in the United States, has an excellent record of reliability. The IR laser it uses is designed for a longer lifetime than the UV laser used by the PALM and Leica estimated at 2 years or 2,000,000 pulses. The PALM, now marketed and supported by Zeiss, and Leica are expanding their operations in the United States, relying for applications and service on the preexisting networks of the two respective companies. We have tested all three systems in an attempt to find the one that best fits most of our needs. Our experience showed that for all three systems, sample preparation and environmental factors are critical for good dissection. Specimens freshly prepared according to the Arcturus-suggested protocols could be very easily dissected. Samples insufficiently dried or stored without desiccation could not be dissected. Specimens prepared for demonstration by the PALM could be easily dissected and catapulted. With a specimen that we prepared with membrane coated slides provided by PALM which maintained a little humidity, the membrane could not be cut reliably even with the highest setting for the laser energy. When the tissue was mounted directly on a glass slide without a membrane, individual cells could be easily catapulted into the collecting cap.

[1]

COMPARISON OF CURRENT EQUIPMENT

11

Dissection using the Leica A S L M D system was less sensitive to sample preparation. Tissue sections were mounted in this case on a thin PEN membrane held by a plastic frame the size of a microscope slide that was easy to dry. Dissected samples, however, were subjected to strong electrostatic attraction from the many charged plastic and metal surfaces close by, allowing only few of them to be collected in the caps. Air humidity in this case is helpful in reducing the electrostatic charges around the membrane. Shooting the slide with an electrostatic gun also helped to some degree. The ability to visually inspect the dissected material was excellent for the Arcturus system. The images of the remaining and dissected material, acquired with the same objective, match each other perfectly and may be added to reconstitute the image before the dissection. In the case of the PALM, larger areas dissected could be visually inspected with relative ease, albeit with a lower magnification objective. Cells mounted directly on the glass slide, dissected by catapulting, are practically pulverized and therefore cannot be seen. Dissected cells could not be seen in the Leica AS LMD. For each instrument we tested the presence and integrity o f R N A in the collecting caps. Groups of cells or single cells were captured with each microscope. Cells were snap-frozen immediately after capture. R N A was extracted using the Arcturus PicoPure R N A Isolation kit following the Arcturus protocol. Extracted R N A was dried down to 1.6 /~l in a vacuum centrifuge. The following components were added to each sample: 1.7/~l Invitrogen 1st Strand Buffer, 0.5/_,1 RNasin (Promega), 1.7/~l 0.1 M D T T (Invitrogen), 1 ~ l dNTPs (Promega), 100 ng Random Hexamers (Invitrogen), and 1 /~1 Superscript II Reverse Transcriptase (Invitrogen) to a final volume of 8.5/~l. The samples were incubated at 42 ° for 90 min.

FIG.6. All captured samples contain intact mRNA. Laser-captured samples were subjected to RNA extraction with the Arcturus PicoPure kit and reverse transcriptionwith the GIBCO-BRLSuperscript II enzyme. Two rounds of PCR were performed with nested primers recognizing cyclophilin, a housekeeping gene. Lanes 1 and 2 contain a sample isolated with the PALM equipment, lanes 3 through 8 contain samples isolated with the Leica AS LMD, and lanes 9 and 10 contain a sample isolated with the Arcturus PixCell system. Lanes 11 and 12 contain PCR products from control rat hypothalamic mRNA. Lanes 1, 3, 5, 7, 9, and 11 represent the first round of nested PCR using outer primers. Lanes 2, 4, 6, 8, 10, and 12 represent the final PCR product after a second round of PCR with inner primers.

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[2]

TABLE I COMPARISON OF CURRENT EQUIPMENT PixCell II

PALM

Leica LMD

+++ ++ + ++ +++ +++ ++ +q-q++ +++ ++

+ +++ +++ +++ ++ ++ +++ + ++ +++ +

+ +++ ++ +++ ++ ++ +++

Laser lifetime Resolution Versatility Sample preparation Sample preparation protocols Ease of use--single cells Ease of use--larger areas Visualization of dissected sample Sample recovery RNA integrity Price

+ +++ ++

After the reverse transcription reaction, 1 ]zl from each sample was subjected to two 35-cycle rounds of PCR with nested primers recognizing cyclophilin, a housekeeping gene. The PCR products from both rounds were electrophoresed on a 2% agarose gel (Fig. 6). Our experience with the instruments tested is summarized in Table I.

Addendum: Useful Sites

http://www.arctur.com/ http://www.palm.spacenet.de/ http://www.leica-microsystems.com/

[2] Laser Capture Microdissection and Its Applications in Genomics and Proteomics By JAMES L. WITTLIFF and MARK G. ERLANDER Background Human tissue collection, handling, and analyses present specific problems for clinically reliable genomic and proteomic testing unlike studies with animal tissues or homogeneous cell lines grown in culture. For example, determinations of levels of clinically relevant analytes in tissue biopsies, used as markers for detection, diagnosis, prognosis, or therapeutic response of a cancer patient, are performed

METHODSIN ENZYMOLOGY,VOL.356

Copyright2002,ElsevierScience(USA). Allfightsreserved. 0076-6879/02$35.00

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BASIC PRINCIPLES

[2]

TABLE I COMPARISON OF CURRENT EQUIPMENT PixCell II

PALM

Leica LMD

+++ ++ + ++ +++ +++ ++ +q-q++ +++ ++

+ +++ +++ +++ ++ ++ +++ + ++ +++ +

+ +++ ++ +++ ++ ++ +++

Laser lifetime Resolution Versatility Sample preparation Sample preparation protocols Ease of use--single cells Ease of use--larger areas Visualization of dissected sample Sample recovery RNA integrity Price

+ +++ ++

After the reverse transcription reaction, 1 ]zl from each sample was subjected to two 35-cycle rounds of PCR with nested primers recognizing cyclophilin, a housekeeping gene. The PCR products from both rounds were electrophoresed on a 2% agarose gel (Fig. 6). Our experience with the instruments tested is summarized in Table I.

Addendum: Useful Sites

http://www.arctur.com/ http://www.palm.spacenet.de/ http://www.leica-microsystems.com/

[2] Laser Capture Microdissection and Its Applications in Genomics and Proteomics By JAMES L. WITTLIFF and MARK G. ERLANDER Background Human tissue collection, handling, and analyses present specific problems for clinically reliable genomic and proteomic testing unlike studies with animal tissues or homogeneous cell lines grown in culture. For example, determinations of levels of clinically relevant analytes in tissue biopsies, used as markers for detection, diagnosis, prognosis, or therapeutic response of a cancer patient, are performed


Copyright2002,ElsevierScience(USA). Allfightsreserved. 0076-6879/02$35.00

[9,]

LASER CAPTURE MICRODISSECTION

13

either biochemically or by immunohistochemistry currently (e.g., Wittliff et al.1). If the analyte (e.g., estrogen receptor, HER-2/neu oncoprotein) is measured biochemically, a tissue specimen consisting of a heterogeneous cell population is homogenized and the final concentration of the analyte extracted from the cancer cells is "diluted" by the contribution of other proteins released from noncancerous cells (e.g., epithelium, histiocytes, macrophages, and connective tissue cells). Therefore, an underestimate of the analyte concentration is likely to be determined compromising the appropriate cutoff value between disease and normal states. While certain tumor markers in tissue biopsies have well served the clinical management of cancer patients (e.g., estrogen receptors in the selection of tamoxifen-responsive breast cancerl), many questions of analyte expression in normal and neoplastic cells remain. Likewise, immunohistochemistry is used to measure certain proteins in cancer tissue sections for clinical application in spite of reports indicating the results are often highly operator and antibody dependent and, at best, semiquantitative (e.g., Igarashi et al.2). As Wittliff 1 and Cole etal. 3 have noted, collection and processing of human tissue biopsies have focused on their clinical purpose (e.g., diagnosis, staging, prognosis, therapy selection) with little emphasis on sampling and cryopreservation for sophisticated genomic (e.g., microarrays) and proteomic analyses (e.g., protein chips). The obvious problem of cellular heterogeneity in the tissue section, which may result in misleading or confusing molecular findings, 3 complicates these issues. Therefore, a reproducible method for obtaining homogeneous cell populations from normal tissue or from cancer biopsies was required in order to obtain accurate information from molecular analyses. Laser capture microdissection (LCM) was initially conceived by a team of investigators at the National Institutes of Health, led by Lance Liotta, Robert Bonner, and Michael Emmert-Buck, to address this need. 4,5 LCM provides a rapid and direct method for procuring homogeneous subpopulations of cells or complex structures for biochemical and molecular biological analyses. Arcturus Engineering, Inc. (Mountain View, CA) developed the first commercial LCM instrument, made available in 1997, in collaboration with the NIH group as part of a Cooperative Research and Development Agreement (CRADA). l j. L. Wittliff, R. Pasic, and K. I. Bland, in "The Breast: ComprehensiveManagementof Benign and Malignant Diseases" (K. I. Bland and E. M. Copeland III, eds.), p. 458. W. B. Saunders Co., Philadelphia, 1998. 2 H. Igarashi,H. Sugimura, K. Maruyama,Y. Kitayama,I. Ohta, M. Suzuki, M. Tanaka,Y. Dobashi, and I. Kino,APMIS 102, 295 (1994). 3 K. A. Cole, D. B. Krizman,and M. R. Emmert-Buck,Nat. Genet. 21, 38 (1999). 4 M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 5 R. E Bonner, M. R. Emmert-Buck,K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997).

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BASIC PRINCIPLES

[2]

FIG. 1. Components of a laser capture microdissection instrument. The LCM station integrates a research grade inverted microscope, a low-power infrared laser, a joystick-controlled stage, and a custom CapSure LCM cap handling mechanism (cassette module and placement ann). The LCM employs a video camera connected to an image archiving unit (not shown) for annotation, storage, and review of the microdissection process.

Laser Capture Microdissection Instrumentation LCM represents a major advancement in nondestructive cell sampling technology that can be applied to genomic and proteomic studies. Studies conducted in our laboratories utilize the PixCell II LCM System (Arcturus Engineering, Inc.) composed of the LCM instrument with fluorescence microscopy, the CapSure Transfer Film Carder, and the PixCell II Image Archiving Workstation (Fig. 1). Briefly, the LCM station integrates a research-grade inverted microscope, a lowpower infrared laser, a joystick-controlled stage, and a custom CapSure LCM cap handling mechanism with a video monitor and controller. Protocol for P r o c e s s i n g H u m a n T i s s u e S p e c i m e n s To evaluate differences between normal and diseased cells, one must first isolate the cells or structures by LCM (Fig. 2) and extract them independently for DNA, RNA, or protein analyses.6,7 Proper tissue procurement, specimen handling, and cryopreservation are essential for the collection of quality information from these analyses) Briefly, biopsy specimens should be excised expeditiously and without trauma during the surgical procedure. Specimens must be chilled on ice, 6 N. L. Simone, R. E Bonner, J. W. Gillespie, M. R. Emmert-Buck, and L. A. Liotta, Trends Genet. 14, 272 (1998). 7 j. L. Wittliff, S. T. Kunitake, S. S. Chu, and J. C. Travis, J. Clin. Ligand Assay 23, 66 (2000).

[2]


15

Removal of Tissue Biopsy Cryopreservation Preparation of Frozen Tissue Sections Evaluate Integrity of Macromolecules Collection of Frozen Tissue Sections on Microscope Slides without Cover Slips Fixation, Staining & Dehydration Laser Capture Microdissection of Normal & Neoplastic Cells or Structures Collection on CapSure® HS Transfer Film Extraction of Macromolecules using ExtrecSure® Device Perform Analyses of DNA, RNA or Protein FIG.2. Sequence for LCM procurement of cells from a complex tissue section. After tissue collection, fixation, staining, and dehydration as described in the text, cells of interest are located and the CapSure optically transparent cap is placed on the tissue. A laser pulse releases the cell from surrounding structures transferring it to the thermoplastic film. The intact cell bound to the CapSure device is lifted and placed onto a standard 500/zl microcentrifuge tube for subsequent extraction and analysis. and then well trimmed of necrotic tissue, leaving normal tissue present with the lesion in question. The tissue specimen should either be frozen on dry ice in the pathology suite within 2 0 - 3 0 min of collection or rapidly transported chilled in a petri dish or plastic bag immersed in ice prior to cryopreservation and frozen section preparation in the L C M laboratory to retain the biological integrity of macromolecules. Any procedure avoiding RNase contact is desirable. It is preferable to freeze the specimen immediately on dry ice after collection at the time of frozen section diagnosis if studies requiring R N A are to be conducted. With the advent o f L C M and sensitive technologies o f genomics and proteomics requiting nondestructive isolation of pure cell populations, new surgical pathology

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BASIC PRINCIPLES

[2]

approaches and methods must be developed as recommended by Cole et al. 3 and Wittliff et al. 7 Specimens are processed according to accepted biohazard policies in clean rooms prepared to reduce RNase contamination. Specimens are frozen in an optimum cutting temperature compound (TissueTek OCT medium, VWR Scientific Products Corp.) and stored at - 8 6 ° until LCM is performed. At that time, frozen sections are collected on sterile microscope slides without a coverslip and retained frozen by being placed on a flat surface of dry ice to preserve labile macromolecules. We recommend that glass slides without coatings (uncharged) be used to enhance LCM of selected cells. Frozen tissue biopsies or tissue sections collected on slides and stored in sterile plastic slide holders may be shipped to a distant laboratory for LCM analyses if the specimens are retained on dry ice during transfer. Preservation of the biological integrity of the biopsy tissue prior to arrival in the LCM laboratory is the shared responsibility of the pathologist and the surgeon, if proteomic and genomic analyses are to become routine clinical tests. In addition, sections of the tissue procured must be representative of the lesion. Fixation, Staining, and Dehydration Frozen sections mounted on uncoated glass slides are handled according to the following procedures depending on the type of staining reagent used. The intercalating dye, TO-PRO-3 (Molecular Probes, Inc., Eugene, OR), which binds tightly to double-stranded nucleic acids and exhibits a peak fluorescence at 661 nm, has been used to assess the integrity of DNA in LCM procured cells and structures. 7 TO-PRO-3 Staining Protocol

(1) Place frozen section in 70% ethanol for 1 min, (2) transfer to PBS for 30 sec, (3) place slide in tray and stain with 10/~M TO-PRO-3 for 2 min, (4) transfer to PBS for 2 min, (5) transfer to deionized water for 30 sec, (6) transfer to 70% ethanol for 30 sec, (7) transfer to 95% ethanol for 30 sec, (8) transfer to 100% ethanol for 30 sec, (9) transfer to xylene for 5 min, and (10) air dry for 20 min; store desiccated. Phosphate-buffered saline (PBS) and deionized water are used in all wash steps. All of the steps utilizing ethanol employed ethyl alcohol UPS (Aaper Alcohol and Chemical Co., Shelbyville, KY). H & E Staining Protocol

(1) Place frozen section in 70% ethanol for 1 min, (2) transfer to hematoxylin Gill No. 3 (Sigma Diagnostics, St. Louis, MO) for 30 sec, (3) transfer to RNasefree water for 30 see, (4) transfer to bluing agent (ThermoShandon, Pittsburgh, PA) for 30 see, (5) transfer to 70% ethanol for 30 see, (6) transfer to 70% alcohol-based eosin Y alcoholic (ThermoShandon, Pittsburgh, PA) for 30 sec, (7) transfer to 70%

[2]

LASER CAPTUREMICRODISSECTION

17

FIG. 3. Representativeimages of human breast carcinomas stained with H & E. (A) Typicalhuman breast carcinomashowinginfiltration of cancer cells into stromal elements; (B) breast carcinomaadjacent to a large population of inflammatorycells; (C) tissue specimenof DCIS with protein secretions; (D) breast carcinomabiopsy exhibiting freezing artifact.

ethanol for 30 sec, (8) transfer to 95% ethanol for 30 sec, (9) transfer to 100% ethanol for 30 sec, (10) transfer to xylene GR/ACS (EM Science, Gibbstown, NJ) for 5 min, and (11) air dry for 20 min; store desiccated. Desiccate only if slide will not be used for RNA extraction. Tissue slides to be used for total RNA extraction and gene expression profiling must be used within 1-2 hr for LCM procurement of cells. Prior to LCM, we evaluate the structural status of the frozen tissue biopsy after sectioning and H & E staining (Fig. 3). As illustrated in Fig. 3A, the section indicates the biopsy is acceptable for proceeding with LCM and gene expression profiles. The section shown in Fig. 3B also indicates an acceptable specimen but considerable caution must be exercised to avoid removing unwanted inflammatory cells with carcinoma cells. The tissue section shown in Fig. 3C illustrates that the specimen received in the laboratory contained considerable areas of DCIS although

18

BASIC PRINCIPLES

[21

the diagnosis indicated invasive ductal carcinoma. This illustrates the value of pathology confirmation on the portion of the tissue biopsy received in the LCM laboratory. Finally, the tissue section shown in Fig. 3D depicts significant freezing artifact indicating the biopsy was unsatisfactory for LCM and gene expression profiling. Prior to LCM, other types of tissue preparations were utilized. These include either formalin-fixed or alcohol-fixed sections that are paraffin-embedded, as well as cytospin preparations of cells from blood or ascites fluids. Fixation conditions are dictated by the nature of the antigen of interest. Precipitative reagents such as acetone and methanol are used for intracellular antigens while cross-linking fixatives such as glutaraldehyde or paraformaldehyde are used for cell-surface antigens. 8 Immunohistochemistry of protein analytes has been performed to guide cell selections by LCM (e.g., Fend et al.9). We routinely perform immunohistochemistry of clinically relevant analytes such as estrogen and progestin receptors, EGF receptors, and HER-2/neu oncoprotein to direct the procurement of cells expressing particular tumor markers.1 S t e p s in L a s e r C a p t u r e M i c r o d i s s e c t i o n The sequence of tissue collection, cell procurement by LCM, and macromolecular extraction is depicted in Fig. 4. Avoid the presence of moisture (e.g., frost, fingertips, breath, room humidity) during all steps prior to RNA extraction. Because our LCM laboratories are used for proteomic and gene expression studies, all procedures are conducted under RNase-free conditions, including cleaning of the stage and related areas of the LCM instrument and surrounding bench with RNase AWAY (Molecular BioProducts, San Diego, CA). Gloves and lab coats are worn at all times. Prior to performing LCM, the joystick should be positioned perpendicular to the bench and the CapSure LCM cap should be placed over the tissue under examination. The operator locates the cell or structure to be microdissected from the tissue section by viewing the histology on the monitor of the PixCell II LCM System. 7 After test firing the IR laser in an area devoid of cells and observing the features of the melted plastic ring, the settings for power and duration are adjusted to obtain the desired spot size. Typically if one is using CapSure HS LCM caps, the following adjustments are suggested: spot size of 7.5/zm (power setting = 65-75 roW; duration setting--650-750 /zs), spot size of 15 /zm (power setting = 35-45 mW; duration setting = 2.5-3.0 ms), spot size of 30 # m (power 8 S.-R. Shi, J. Gu, and C. R. Taylor, "Antigen Retrieval Techniques: lmmunohistochemistry and Molecular Morphology." Eaton Publishing, Natick, MA, 2000. 9 E Fend, M. R. Emmert-Buck, R. Chuaqui, K. Cole, J. Lee, L. A. Liotta, and M. Raffeld, Am. J. Pathol. 1S4, 61 (1999).

[2]


19

O

! FIG. 4. Simple four-step process to capture cells and recover macromolecules. After location of the cells of interest, a CapSure or CapSure HS LCM cap is placed over the target area. Pulsing the laser through the cap activates the thermoplastic film to form a thin protrusion that bridges the gap between the cap and tissue and adheres to the target cell. Lifting of the cap removes the target cell(s) now attached to the cap. Macromolecules may be extracted from 200-1000 cells using the ExtracSure Sample Extraction Device which accommodates small volumes.

setting = 4 5 - 5 5 m W ; duration setting = 6 . 0 - 7 . 0 ms). I f C a p S u r e L C M caps are e m p l o y e d , the f o l l o w i n g adjustments are suggested: spot size o f 7.5 /zm (power setting = 4 0 - 5 0 m W ; duration setting = 5 5 0 - 6 5 0 /zs), spot size o f 15 # m (power setting = 3 0 - 4 0 m W ; duration setting = 1.5-2.0 ms), spot size o f 3 0 / z m (power setting = 2 5 - 3 5 m W ; duration setting = 5 . 0 - 6 . 0 ms). In order to correctly return to the initial area o f cell capture for m a k i n g photo records o f

20

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[21

"After" and "Cap" images (Fig. 5), the authors "mark" the region by placing several spots on areas devoid of cells adjacent to the cells of interest. This is particularly helpful when collecting multiple caps of cells from distant regions in the same tissue section for later comparison of proteomic and genomic analyses. Using the image archiving unit, characteristics of the tissue section are recorded before and after LCM, as well as those of the cells procured on each cap (Fig. 5). The cells or structures are microdissected after firing the IR laser and lifting the CapSure LCM cap with the intact cells collected on the transfer film. The CapSure and CapSure HS consist of a proprietary thermoplastic polymer film hermetically sealed to the bottom of a precision optical grade plastic cap. In certain experiments requiring extraction of small numbers of cells (200-1000) in low microliter volumes, we utilize the ExtracSure Sample Extraction Device (Arcturus) and the CapSure HS LCM caps for efficient removal of total RNA. The CapSure LCM caps containing the cells fit directly onto standard reagent tubes (500/zl Eppendorf) in preparation for cell extraction. Typically, 1-6 ng of total RNA may be extracted in this manner using Buffer RLT (Qiagen, Valencia, CA). The transfer process does not damage the captured cells or the surrounding cells remaining on the slide containing the original tissue preparation (Fig. 5). Usually there is no undesirable cellular contamination since the IR laser beam may be focused between 7.5 and 30/zm providing accurate selection. If necessary, we employ the CapSure pads to remove debris (e.g., stromal elements) from the CapSure LCM caps prior to extraction. Forces involved in an efficient LCM manipulation include (a) those between tissue and slide, (b) those between tissue and activated film, (c) tissue-tissue interactive forces, and (d) the force between tissue and inactivated film. The dynamics of the IR focusing and the melting properties of the thermoplastic transfer film on the CapSure LCM caps are optimized with those of cells in 5- to 10-/zm tissue sections. After collection of cells on the CapSure LCM cap, macromolecules are extracted using a variety of procedures depending on whether the analyses are focused on DNA, RNA, or protein, as described in other chapters of this volume. Gene expression as measured by analyses of mRNA provides an understanding of the manner in which normal cells respond to endocrine changes, malignant transformation, and environmental insults. 6"7"1°-13Determination of the level of gene 10 A. Glasow, A. Haidan, U. Hilbers, M. Breidert, J. Gillespie, W. A. Scherbaum, G. E Chrousos, and S. R. Bornstein, J. Clin. Endocrin. Metab. 83, 4459 (1998). l l L. Luo, R. C. Salunga, H. Guo, A. Bhittner, K. C. Joy, J. E. Galindo, H. Xiao, K. E. Rogers, J. S. Wan, M. R. Jackson, and M. G. Erlander, Nat. Med. 5, 117 (1999). 12 D. C. SgToi, S. Teng, G. Robinson, R. LeVangie, J. R. Hudson, Jr., and A. G. Elkahloun, Cancer Res. 59, 5656 (1999). 13 S. M. Goldsworthy, E S. Stockton, C. S. Trempus, J. E Foley, and R. R. Maronpot, Mol. Carcinog. 25, 86 (1999).

[2]


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r

FIG. 5. Representative collection of human breast cancer cells by LCM. Three different regions of the same breast carcinoma biopsy are shown in the top three panels marked Before. The regions of the tissue section where carcinoma cells were removed by LCM are shown in the images marked After, and the isolated cells adhering to the CapSure LCM caps are shown in the images marked Capture, Each cap, containing 200-300 carcinoma cells, is extracted for RNA that is quantified and amplified before microarray analyses.

expression as well as the size and structure of RNA molecules requires retention of biological integrity. Because of the lability of mRNA, several workers have studied the effects of tissue fixation on RNA extraction and amplification after LCM, 9,13 providing some insight into the stability of these labile molecules using current procedures. A d v a n t a g e s of LCM Manual microdissection techniques, which require tedious manipulation, significant manual dexterity, and a lengthy training program, are slow and the variability in tissue collection is significant. However, LCM, which uses standardized

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[2]

technology, allows rapid sample procurement of tissue structures with awkward geometry and efficient isolation of different cell types in close proximity or adjacent to each other. 2-4,7 Furthermore, the transfer process is nondestructive and cell morphology is retained.7 Of particular importance in molecular diagnostics and gene discovery, there is a record of the original location of cells in the tissue and visual verification of cell capture. Some investigators have reported successful DNA analyses using 300-500 cells (e.g., Simone et al.,6; Sirivatanaukorn et al.14), while 500-1000 cells have been used to isolate RNA (e. g., Glasow et al. 10; Luo et al. 11; Goldsworthy et al. 13). Examinations of proteins using a single technology have employed 10005000 cells isolated by LCM (e.g., Banks et al)5; Emmert-Buck et al.16). Extraction and 2D PAGE of proteins from representative samples requires capturing 20,000-30,000 cells although new nanotechnology approaches are being developed. 15,16 RNA Isolation, C h a r a c t e r i z a t i o n , a n d A m p l i f i c a t i o n for M i c r o a r r a y In our laboratories, total RNA is isolated using the PicoPure (Arcturus) kits, which are optimized for extracting RNA from cells procured by LCM. Routinely I-6 ng of total RNA may be isolated from 200--300 human breast cancer cells procured by LCM, using these reagents. The intactness of RNA in tissue sections is evaluated prior to proceeding with LCM by a variety of procedures including electrophoresis incorporating a series of markers of different base-pair lengths. For investigations of gene expression profiles of human tissues, we procure cells of interest (e.g., normal vs neoplastic) from at least three different regions of a single tissue section (Fig. 5). Note that the carcinoma cells were removed from each of the three regions of interest and procured cleanly and retained on the CapSure LCM caps (Fig. 5, lower images). Each cell capture (usually containing 200-I000 cells) is treated as an independent evaluation in that the RNA is extracted, purified, and amplified, then subjected to microarray (Pig. 6). RNA isolated from cells procured by LCM is amplified efficiently with the RiboAMP kits (Arcturus) enabling the production of microgram amounts of RNA from nanogram quantities isolated from breast carcinoma and normal cells. Amplification requires preparation of double-stranded cDNA from the mRNA fraction of total RNA followed by transcription in vitro. The use of exogenous primers maximizes reliability in the synthesis of cDNA template while reducing reaction 14y. Sirivatanaukom, V. Sirivatananksorn, S. Bhattacharya, B. R. Davidson, A. P. Dhillon, A. K. Kakkar, R. C. N. Williamson, and N. R. Lemoine, I. Pathol. 189, 344 (1999). 15 R. E. Banks, M. J. Dunn, M. A. Forbes, A. Stanley, D. Pappin, T. Naven, M. Gough, P. Harnden, and P. L Selby, Electrophoresis 20, 689 (1999). 16 M. R. Emmert-Buck, J. W. Gillespie, C. P. Paweletz, D. K. Ornstein, V. Basrur, E. Appella, Q. H. Wang, J. Huang, N. Hu. P. Taylor, and E. E Petricoin III, Mol. Carcinog. 27, 158 (2000).

[2]


23

CaEo8 ER-

I

G E N E S

I;D~

FIG. 6. A representative Eisen Color Map of the gene expression profiles of various human breast carcinomas. Although the microarray performed contained more than 12,000 genes, only a portion of the gene expression profile of each breast cancer is shown using the GeneMaths program (Applied Maths, Austin, TX). Note that without preconceived selection of criteria, gene clustering was observed. Through preliminary bioinformatic analyses, molecular signatures are being identified for several types of human breast cancers, such as those expressing estrogen receptors (ER+) compared with carcinomas lacking the receptor (ER-), which is a marker of anti-estrogen responsiveness. Principal component analysis (diagram on right) was performed using the data matrix shown on the left, and the collective results of the breast specimens are projected onto the three-dimensional space diagram using the first three components.

times. The a R N A prepared b y this protocol is ready for labeling and hybridization necessary for microarray analyses. Preliminary studies of microarray analyses of i n d e p e n d e n t amplifications from the same R N A preparation indicate an excellent correlation. RT-PCR was used to detect low-, m e d i u m - , and h i g h - a b u n d a n c e genes w i t h i n the amplified R N A population. Our laboratory has demonstrated that

24

BASIC PRINCIPLES

[2]

amplification of mRNA in all abundance classes ensures that differential gene expression patterns will be identified. Currently, we are using a microarray containing approximately 12,000 genes of which 10% were included through KnowledgeBased Selection based on reported alterations in cancer. From studies of more than 100 human breast cancers, we have demonstrated that the RNA isolated from LCM procured cells is intact for use in amplification of mRNA and subsequent microarray (Fig. 6). We are employing this approach to derive molecular signatures (gene expression profiles) to advance the classification of breast cancer and assessment of patient prognosis and therapeutic response. A d d i t i o n a l A p p l i c a t i o n s of L a s e r C a p t u r e M i c r o d i s s e c t i o n LCM is rapidly becoming the method of choice for selecting diseased cells from normal cells of the same tissue specimen for genomic and proteomic analyses. 7'I°-16 Some of the applications of LCM in these areas related to molecular diagnostics and prognostics of human cancer are shown below. Genomics: Differential gene profiling Loss of heterozygosity Micro-satellite instability Gene quantification Mutation/clonal analysis Proteomics: Two-dimensional PAGE Western blots Immunoquantitation of proteins MALDI-TOF mass spectrometry The ability to procure homogeneous cell subpopulations of normal, premalignant, and malignant cell types and to accumulate data from each cell type advances our understanding of the underlying causes of tumor formation and permits the tracking, at the molecular level, of cell progression into a metastatic phenotype. Efforts are well underway to produce cDNA libraries that catalog genes differentially expressed during tumor progression (e.g., Peterson e t a/.17). The Cancer Genome Anatomy Project (CGAP) has utilized LCM to obtain normal and premalignant samples from human prostate, breast, ovary, colon, kidney, and 17L. A. Peterson,M. R. Brown,A. J. Carlisle, E. C. Kohn,L. A. Liotta,M. R. Emmert-Buck,and D. B. Krizman, Cancer Res. 58, 5326 (1998).

[3]

LIVE CELL LASER MICRODISSECTION

25

lung tissue, to name a few. Information from CGAP is publicly available through the CGAP-NIH Web site.18 Laser capture microdissection has proved to be a powerful tool for research into the cellular basis of disease and is increasingly being employed in drug discovery and clinical diagnostics. Physiological changes occurring during development and progression of normal cells to neoplastic lesions may be explored easily with LCM and proteomics and gene expression profiling. For clinical diagnosis, the ability to sample specific types of cells creates a new analytical paradigm which will allow patients to be diagnosed based on qualitative and quantitative gene expression as well as on levels of cell-specific proteins. As Wittliffsuggested previously, 1a new generation of laboratory tests is rapidly evolving in which analyses will be performed directly on human tissue biopsies. It is envisioned that tissue banks such as the Biorepository at the Hormone Receptor Laboratory will be developed for long-term preservation of human tumor samples. This will allow assessment of genetic and biochemical properties of the stored tumor tissues as new clinical, chemical, and molecular biological probes are developed for cancer management, and as technologies such as laser capture microdissection are utilized to separate normal from tumor cells. 18 www.ncbi.nlm.nih.gov/CGAP

[3] G o i n g

in Vivo with Laser Microdissection

By ANETTEM A Y E R , MONIKASTICH,

DIETER BROCKSCH, K A R I N SCHUTZE,

and GEORGIALAHR Introduction Tissue microdissection and single-cell isolation is one of the most advanced techniques in modem gene analysis and is especially useful for studying expression of genes in isolated tumor cells. Till now, microdissection methods have been limited to cells from fixed or frozen tissues. 1-9 An old dream of cell biologists 1 W. Meier-Ruge, W. Bielser, E. Remy, E Hillenkamp, R. Nitsche, and R. Uns~51d,Histochem. J. 8, 387 (1976). 2 M. Schindler, M. L. Allen, M. R. Olinger, and J. E Holland, Cytometry 6, 368 (1985). 3 y. Kubo, E Klimek, Y. Kikuchi, E Bannasch, and O. Hino, CancerRes. 55, 989 (1995). 4 M. R. Emmert-Buck, R. E Bonner, E D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 5 R. E Bonner, M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997).


Copyright2002,ElsevierScience(USA). All rightsreserved. 0076-6879/02$35.00

[3]


25

lung tissue, to name a few. Information from CGAP is publicly available through the CGAP-NIH Web site.18 Laser capture microdissection has proved to be a powerful tool for research into the cellular basis of disease and is increasingly being employed in drug discovery and clinical diagnostics. Physiological changes occurring during development and progression of normal cells to neoplastic lesions may be explored easily with LCM and proteomics and gene expression profiling. For clinical diagnosis, the ability to sample specific types of cells creates a new analytical paradigm which will allow patients to be diagnosed based on qualitative and quantitative gene expression as well as on levels of cell-specific proteins. As Wittliffsuggested previously, 1a new generation of laboratory tests is rapidly evolving in which analyses will be performed directly on human tissue biopsies. It is envisioned that tissue banks such as the Biorepository at the Hormone Receptor Laboratory will be developed for long-term preservation of human tumor samples. This will allow assessment of genetic and biochemical properties of the stored tumor tissues as new clinical, chemical, and molecular biological probes are developed for cancer management, and as technologies such as laser capture microdissection are utilized to separate normal from tumor cells. 18 www.ncbi.nlm.nih.gov/CGAP

[3] G o i n g

in Vivo with Laser Microdissection

By ANETTEM A Y E R , MONIKASTICH,

DIETER BROCKSCH, K A R I N SCHUTZE,

and GEORGIALAHR Introduction Tissue microdissection and single-cell isolation is one of the most advanced techniques in modem gene analysis and is especially useful for studying expression of genes in isolated tumor cells. Till now, microdissection methods have been limited to cells from fixed or frozen tissues. 1-9 An old dream of cell biologists 1 W. Meier-Ruge, W. Bielser, E. Remy, E Hillenkamp, R. Nitsche, and R. Uns~51d,Histochem. J. 8, 387 (1976). 2 M. Schindler, M. L. Allen, M. R. Olinger, and J. E Holland, Cytometry 6, 368 (1985). 3 y. Kubo, E Klimek, Y. Kikuchi, E Bannasch, and O. Hino, CancerRes. 55, 989 (1995). 4 M. R. Emmert-Buck, R. E Bonner, E D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 5 R. E Bonner, M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997).



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BASIC PRINCIPLES

[31

is to isolate living cells from tissue culture or unfixed and unfrozen sections of living tissuesJ ° But to date, laser-based microdissection of living cells resulted in the destruction of the isolated cells. 2 Now, a modification of the laser microbeam microdissection (LMM) method in combination with the laser pressure catapulting (LPC) technique 6 and a newly developed cell culture protocol allows microdissection and "ejection" of living single cells or cell clusters with ongoing cultivation for potential treatment and analysis. We established a unique technique in which cultured cells were microdissected and afterward catapulted by LPC into the cap of a microfuge tube. The viability of the catapulted cells is not affected as they enter the cell cycle and proliferate. Applying this protocol--select, microdissect, eject, and clone living cells--to biopsy slices will come true in the near future. As this, "going in vivo" opens up a broad spectrum of applications. S t e p I: Cell C u l t u r e P r e p a r a t i o n A prerequisite for isolation of single living cells from cell cultures by laserassisted cell picking is the growth of the cells on a supporting membrane. The membrane is mounted in a specific cell culture chamber, the ROC chamber. For microdissection the membrane around the cell or cell clusters of interest is cut by the focused laser beam in a sufficient distance from the cell. Then the cell-membrane stack is catapulted by the laser beam into a conventional cap of a microfuge tube centered directly above the selected area (Fig. 1, A and B). Buffers, Reagents, and Equipment

ROC chamber, Round Open Closed (PeCon and LaCon, Erbach-Bach, Germany) Polyethylene-naphthalene membrane, 1.35/zm (PEN membrane; P.A.L.M. Microlaser Technologies AG, Bemried, Germany) EJ28 cells, a bladder carcinoma cell line TPC-1 cells, a thyroid carcinoma cell line Dulbecco's modified Eagle's medium [Invitrogen GmbH (GIBCO-BRL), Karlsruhe, Germany] Dulbecco's modified Eagle's medium Nutrient Mixture F 12-Ham (DME/F12 Hams) (Sigma-Aldrich GmbH, Deisenhofen, Germany) 200 mM L-glutamine (Sigma-Aldrich GmbH) 6 K. Schiatzeand G. Lahr, Nat. Biotechnol. 16, 737 (1998). 7 K. Schiitze, H. POsl,and G. Lahr, Mol. Cell Biol. 44, 735 (1998). 8 G. Lahr, Lab. Invest. 80, 1 (2000). 9 G. Lahr, M. Stich, K. Sch~tze, P. Bliimel, H. Ptsl, and W. B. J. Nathrath, Pathobiology 68, 218 (2oo0). lOM. Schindler, Nat. Biotechnol. 16, 719 (1998).

[3]


A

27

NOC chamber with cell culture

FIG. 1. Schematic drawings of a cell culture grown on a PEN membrane in an ROC chamber. The chamber is attached to the microscope stage and the microfuge cap is centered above the line of laser fire directly in the ROC chamber (A). The selected cell-membrane stacks are microdissected by the laser beam (LMM). The cell-membranestacks are catapultedby LPC directly into the cap of the sample tube supplied with a droplet of Hanks' solution (B). The captured ceils are coveredwith 25 #1 of Hanks' solution (C). The cap is topped with the remaining tube and the assembledtube is centrifuged to collect captured cells at the bottom of the microfuge tube (D).

10% Fetal calf serum (FCS; Sigma-Aldrich GmbH) 100 x Antibiotic-antimycotic solution (Sigma-Aldrich GmbH) Hanks' solution (Sigma-Aldrich GmbH) T r y p s i n - E D T A solution (Sigma-Aldrich GmbH) Conventional culture dish plates for cell culture Gassed incubator for cell culture Laser microscope, Robot-MicroBeam (EA.L.M. Microlaser Technologies AG) Inverted microscope Axiovert 135 (Carl Zeiss, G6ttingen, Germany) Microfuge tubes (EA.L.M. Microlaser Technologies AG)

Procedure Assembly of the ROC Chamber 1. Cover the glass bottom o f the ROC chamber with the p o l y e t h y l e n e naphthalene membrane (PEN membrane) by using a droplet of 100% ethanol for mounting it onto the glass. 2. Expose the opened chamber with the membrane to UV light for 20 min to change the hydrophobic nature of the membrane into a more hydrophilic one. 3. Assemble the ROC chamber totally and autoclave it at 121 ° for 20 min.

28

BASIC PRINCIPLES

[31

Cell Culture. The EJ28 bladder carcinoma cell line and the papillary thyroid tumor cell line TPC-1 (a generous gift of Dr. B. Mayr, Med. Hochschule Hannover, Germany) were used for the experiments. EJ28 cells were grown in Dulbecco's modified Eagle's medium and TPC-1 cells were grown in Dulbecco's modified Eagle's medium Nutrient Mixture F 12-Ham (DME/F 12 Hams), both supplemented with 5 m M L-glutamine, 10% fetal calf serum (FCS), and 1 x antibioticantimycotic solution. Seed the cell culture cells at the desired density onto the membrane-covered ROC chamber in their appropriate medium. 4. Incubate the cells in the ROC chamber at 37 ° in a gassed incubator. After 1-2 days in culture the cells are ready for microdissection.

Laser Microdissection and Catapulting 5. Remove the medium completely from the ROC chamber before laser microdissection (Figs. 1-4A). 6. Microdissect the desired cell-membrane sample. The parameters concerning laser energy and laser focus during microdissection (LMM) are dependent on the laser microscope system used and have to be optimized before use (Figs. 1A, 2B, 3B, 4C, 5B). 7. A p p l y a 10-/zl droplet of Hanks' solution on top of the selected cells to facilitate LPC. Be careful not to wash away the microdissected specimen.

FIG.2. Images using LMM and LPC to capture 40 EJ28 cells. Cells before microdissection (A), after microdissection (B), cells remaining after LPC (C), catapulted membrane with the cells (D). 11 hr after plating (E), after 1 day (F), after 5 days (G), after 8 days (H), and after 12 days (I). Black arrow: cell filopodium. White arrow: mitotic cell. Dotted line: area to be microdissected. Bar equals 100/zm in A-D, F, and H; 50/zm in E, G, and I. Objective lenses in A-D and F: 20x ; E and I: 40x ; G: 5x;andH: 10×.

[3]


29

FIG. 3. LMM and LPC images of a small TPC- 1 cell cluster (10 cells). The sequence shows cells before LMM (A), after LMM (B), and remaining cell culture after LPC (C). (D) Aggregated cells within the "hanging droplet" 9 hr after collection. 1 day in culture the catapulted and aggregated cells begin to adhere to the bottom of the culture dish (E). Proliferating cells shown after 12 days in culture (F). Dotted line: area to be microdissected. Bar equals 50 # m in A-C; 100/zm in D-F. Objective lenses in A-C: 4 0 x ; D-F: 20×.

8. Pipette a 5-/zl droplet of Hanks' solution into the center of the cap of a microfuge tube and place the cap directly above the selected cells into the ROC chamber (Fig. 1A). 9. Catapult the cell-membrane stack with one single laser shot positioned at the border of the circumscribed membrane. For the catapulting the laser is focused below the microdissected target specimen. 10. Energy settings should be sufficiently high to catapult the microdissected cells with the membrane into a cap (Fig. 1, A and B). Even large cell-membrane stacks (for example 385 × 248/zm) can be catapulted (Fig. 2C).

FIG. 4. Images of an experiment to destroy an "undesired cell" before LMM and LPC of about 18 living F.J28 cells. The sequence shows the cells before microdissection(A), after destruction of one specific cell (B), and after microdissection (C), the remaining cells after LPC (D), and the catapulted membrane with the cells (E). (F) Cells after 12 days in culture. Black arrow: cell destroyed by a precise laser shot. White arrow: ceils on the membrane after catapulting. Dotted line: area to be microdissected. Bar equals 100/xm in A-E; 50 tzm in E Objective lenses in A-E: 20 × ; F: 10 ×.

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BASIC PRINCIPLES

[31

FIG. 5. Images of 5 pooled single EJ28 cells. The sequence shows the cells before LMM (A), after LMM of cell group 1 (B), after LMM of cell groups 1 and 2 (C), after LMM of cell groups 1 to 3 (D). The remaining cells after LPC of cell group 1 (E), 1 and 2 (F), and after LPC of cell group 3 (G). (H) 3 catapulted membranes with cells. Aggregated cells within the "hanging droplet" (I). Dotted lines: areas to be microdissected. Bar equals 100/zm in A-H; 5 0 / z m in I. Objective lenses in A-H: 2 0 x ; I: 4 0 x .

11. After LPC remove the ROC chamber from the microscope stage, take the cap, and inspect the catapulted cells in the cap now fixed within the manipulator (Figs. 2D, 4E, 5H). Notes to Step I a. To reduce the chance of contamination wear gloves during the whole cell culture procedure. Do not keep the cell cultures outside the incubator longer than necessary. In case of several experiments allow cells in the ROC chamber to recover from dryness by adding medium back to the cells. This medium has to be removed totally, otherwise during the cutting process the laser energy will be absorbed by the aqueous solution. This results in local heating of the medium and in visible steam bubbles, which destroy the viable cells. In addition, be aware that after several microdissection events the medium is entering the micro space between the membrane and the glass bottom of the ROC chamber. This makes further microdissection and catapulting more and more difficult and finally impossible. b. With the focused laser beam single cells or cell clusters are precisely separated together with the membrane from their surrounding (Figs 2B, 3B, 4C,

[3]


31

5B-5D) using 20 x (Figs. 2, 4, 5) or 40 x (Fig. 3) objective lenses. Laser circumscription of the cell-membrane stack results in a gap, free of any other biological material, separating the target from its surroundings. 6 The width of the gap is about 3-5/zm, depending on the objective lens and the absorption behavior of the specimen. Where the selected specimen area contains an undesired cell, this cell can be eliminated by a direct laser shot (Fig. 4B). c. Increased laser energy catapults the target specimen into the cap of a microfuge tube. Even large cell-membrane stacks (for example 290 x 369/zm) can be catapulted (Figs. 2D, 4E, 5H). This results in empty patches within the cell culture (Figs. 2C, 3C, 4D, 5G). The laser-catapulted cell-membrane stacks are well preserved and allow direct correlation with their templates in terms of shape, size, and original position (Figs. 2D, 4E, 5H). Microdissection and catapulting of cell clusters in these large sizes takes less than 2 min. The manipulation of single living cells is done within seconds. Laser-assisted isolation is performed with cell clusters of about 10 cells (Fig. 3) and several tens (Figs. 2 and 4), as well as single cells (Fig. 5).

S t e p II: C o l l e c t i o n of C a t a p u l t e d Cells Procedure

1. Cover the catapulted cells in the cap with 25 #1 of Hanks' solution. 2. Close the cap with the remaining tube and store for up to 30 min at room temperature. 3. Centrifuge the tube for 1 min at 8000g and discard the supernatant. 4. Resuspend the cells in 20 #l trypsin-EDTA solution and incubate for 10min at room temperature to detach the cells from the membrane. 5. Centrifuge for 1 min at 8000g. 6. After centrifugation the trypsinized cells (pellet) are resuspended in 15 #1 (2-fold in the same direction (increased or decreased) in the duplicate comparisons are culled and put into a dataset. However, using an arbitrary threshold for fold change (e.g., defining an increase or decrease of >2-fold as significant) means that potentially important and reproducible biological changes will be masked. In addition, fold change is a ratio: probe set intensities only reflect expression differences linearly within a limited range; if either probe set has hybridization intensities outside this range (high or low), the ratio will be skewed. Because of these concerns, we have designed an alternative system for filtering false positives. The system is based on the results of our analysis of signal intensities produced by genes whose expression level was called "changed" in chip-to-chip comparisons of an identical RNA population. 35 All called changes in expression in these same-same comparisons were defined as false positives. The distribution of signal intensities of the false positives in baseline and partner GeneChips was compared to the distribution of signal intensities produced when biologically distinct RNAs were analyzed. The results were used to create a series of look-up tables (LUTs). These LUTs can be used to score transcripts whose expression level is called changed by GeneChip software in comparisons of biologically distinct RNAs. LUT-derived scores range from 0 to 6, with 0 most likely and 6 least likely to represent noise. Eliminating transcripts with LUT scores of 2-fold change threshold. LUTs and software needed to score GeneChip datasets are available (gordonlab.wustl.edu/mills). A n a l y z i n g P r o t e i n s in LCM Cell P o p u l a t i o n s A central challenge to doing protein analysis is efficient extraction of material from LCM cells on the cap. Efficient extraction of 50,000 intestinal epithelial cells yields ~ 10 #g of total protein. A comparison of proteins present in two populations of intestinal cells harvested by LCM can entail two-dimensional gel electrophoresis (2D GE) followed by immunoblotting with antibodies directed against a protein of interest. Alternatively, gels can be stained with a sensitive dye, and the relative abundance of a protein or proteins compared in the two cellular populations. Protocol f o r Protein Isolation and 2-Dimensional Gel Electrophoresis

1. Lyse captured cells on a single cap by adding 20 /zl of a solution containing 7 M urea, 2 M thiourea, 4% CHAPS (Sigma Chemical Co.), 40 mM 35j. Mills and J. I. Gordon,Nucleic Acids Res. 29, el3 (2001).

[1 5]

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191

Tris-base, 50 mM DTT, 1% decanoyl-N-methlyglucamide (Sigma), 1% n-octylfl-D-glucopyranoside (Sigma), 2 mM tributylphosphine (Bio-Rad), and 0.5% ampholytes (Bio-Rad). 36 This buffer extracts total cellular proteins. The extracted proteins can be directly analyzed with 2D GE. 2. First dimension isoelectric focusing is carried out using 7-cm-long Ready Strips IPG Strips with a pH 3-10 gradient (Bio-Rad). The strips are equilibrated in two buffers (2 washes of 10 min each): (a) buffer 1 (6 M urea, 2% SDS, 0.375 M Tris-HC1, pH 8.8, 20% glycerol, 130 mM DTT) followed by (b) buffer 2 [6 M urea, 2% SDS, 0.375 M Tris-HC1, pH 8.8, 20% glycerol, 135 mM iodoacetamide (Sigma)]. 3. For second dimension separation, equilibrated strips are applied to a 4-12% NuPage precast Bis-Tris gradient gel (Invitrogen). Electrophoresis is performed in MES buffer (50 mM MES, 50 mM Tris-base, pH 7.3, 3.5 mM SDS, 1 mM EDTA) at 80 volts for 15 min followed by 150-200 volts for 1 hr. The gels can be either electrophoretically transferred to PVDF membranes (Millipore) for immunoblotting, or treated with SYPRO Ruby IEF gel stain (Molecular Probes). SYPRO Ruby can detect as little as 1 ng of protein and has a dynamic range of 1-1000 ng. 37 An approach developed by Cravatt and co-workers may be particularly useful for analysis of the proteomes of laser capture microdissected cells. It is based on changes in protein activity rather than abundance and uses labeled chemical probes directed at active sites (e.g., serine hydrolases). 38 They have also generated combinatorial libraries of electrophilic probes to proteomes: these probes are incubated with cellular proteins prior to electrophoretic separation. A control incubation is performed at a nonphysiological temperature so that heat-sensitive interactions can be distinguished from heat-insensitive (nonspecific) interactions. Reactive protein species exhibiting heat-sensitive interactions are then excised for mass spectrometry-based identification. The approach yields workable sized signatures of enzyme/protein activities.39 A s s a y i n g E n z y m e s a n d M e t a b o l i t e s Involved in I n t e r m e d i a r y M e t a b o l i s m We have developed a method for performing LCM under conditions that preserve cellular metabolites and enzyme activity for subsequent quantitative biochemical assays. This requires an embedding medium that does not interfere with 36 D. K. Ornstein, J. W. Gillespie, C. E Paweletz, E H. Duray, J. Herring, C. D. Vocke, S. L. Topalian, D. G. Bostwick, W. M. Linehan, E. E Petricoin, and M. R. Emmert-Buck, Electrophoresis 21, 2235

(2000). 37 W. F. Patton, Electrophoresis 21, 1123 (2000). 38 y. Liu, M. P. Patricelli, and B. E Cravatt, Proc. Natl. Acad. Sci. U.S.A. 96, 14694 (1999). 39 G. C. Adam, B. E Cravatt, and E. J. Sorensen, Chem. Biol. 8, 81 (2001).

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SPECIALIZEDUSES

[ 15]

enzyme/metabolite measurements or with laser capture microdissection of unstained, unfixed tissue sections. In our method, OCT is replaced with a rabbit brain paste and the sectioned tissue is affixed to the glass slide by freeze-drying.

Tissue Preparation and Sectioning Frozen, stripped rabbit brains (Pel-Freez) are thawed and then disrupted, without added water or buffer, using a high shear tissue homogenizer (Tekmar) set at full power for 3 min. The resulting brain paste should be smooth and free of lumps. An 8- to 10-cm-long specimen of intestine is rapidly harvested as described above, flushed with PBS, and perfused with brain paste. The perfused specimen is subdivided into three "-~2.5 cm segments which are then placed at the base of a TissueTek cryomold. The segments are overlaid with additional brain paste and the cryomold immediately submerged in liquefied Cryocool. Sections (7/zm) are cut in a cryostat cooled to - 1 0 °. Superfrost/Plus glass slides are precooled on dry ice before the tissue section is applied. Slides containing the tissue section are then immediately dropped into liquid nitrogen and freeze-dried at - 3 5 ° under vacuum (0.01 mm Hg) for 2 days. Slides can be stored at - 8 0 ° under vacuum for at least 1 month.

Preparation of Cell Lysates for Enzyme Assays For descriptions of the equipment used to obtain nanoliter-sized aliquots and for other aspects of the cycling assays, see Passoneau and Lowry (1993). 40 Approximately 3000 epithelial cells (50-70 ng soluble protein), or equivalent amounts of mesenchyme or muscle, are collected per cap. Lysates are prepared by adding 1.6/zl of extraction buffer to the surface of the cap [extraction buffer contains 20 mM phosphate, pH 7.4, 5 mM 2-mercaptoethanol, 25% glycerol, 0.5% Triton X-100, 1 m M Pefabloc (Roche) plus 1 tablet of"complete mini-EDTA-free protease inhibitors" (Roche) dissolved in 5 ml of the buffer]. Following a 1 min incubation at 20 °, lysates are removed from the cap surface and transferred to an oil well (a Teflon block containing drilled wells filled with a mixture of mineral oil and hexadecane to prevent evaporation and to facilitate long-term storage). The Teflon block containing the lysates can be stored at - 8 0 ° under vacuum indefinitely. Aliquots of the lysates are removed for assays of enzyme activities as well as determination of soluble protein. Levels of enzymes and metabolites are expressed per microgram of soluble cellular protein. Soluble protein concentration in lysates is defined using the colloidal gold method (Diversified Biotech). A 0.2-#1 aliquot of lysate is added to 0.5 ml colloidal gold reagent. Following a 40j. W.Passoneau,and O. H. Lowry,"EnzymaticAnalysis:A PracticalGuide."HumanaPress, Totowa, NJ, 1993.

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NAD+ Malate_ Ethanol Malic dehydrogenase~ ~ A I c o h o l dehydrogenase Oxaloacetate

NADH

Acetylaldehyde

SCHEMEI. NAD cycle.

Glutamate..Ir~ ~rNADP~.. f Glutamate dehydrogenase "y" )(" ../'~

c(-Ketoglutarate+ NH4+

~

~

NADPH

G-6-phosphate Glucose6-phosphate aenyarogenase

6-Phosphogluconate

SCHEMEII. NADPcycle. 60 min incubation at 37 °, absorbance is determined at 595 nm. Typically, BSA standards are surveyed in the range of 1-10 ng.

Enzyme Cycling: General Principles This sensitive, versatile, and well-established analytic method allows the levels of enzymes, metabolites, and nucleotides to be measured in laser captured cell populations through reactions that generate reduced or oxidized forms of NAD or NADP. The usefulness of using NAD and NADP for analytic purposes has been described by Passoneau and Lowry.40 The low levels of some enzyme activities in LCM cell lysates require that NAD or NADP generated in the primary analytic reaction be amplified through a series of cycling steps. Each cycling step involves one of the following coupled reactions (see Schemes I and II). After an appropriate number of cycles are performed (see Ref. 41 for details about how to determine the number of cycles), the reaction is terminated by heating at 100° for 5 min. Samples are cooled to room temperature, and 1 ml of indicator reagent is added to convert the cycled product (malate in the case of NAD cycling, 6-phosphogluconate in the case of the NADP cycling) to NADH or NADPH, respectively. The NAD cycling indicator step (see Scheme III) involves addition of 1 ml of malate reagent [50 mM aminomethylpropanol, pH 9.9, 5 mM L-glutamate, pH 9.9, 0.2 mM NAD +, 5/zg/ml malic dehydrogenase (3000 U/mg protein, Sigma Chemical Co.), and 2/zg/ml glutamate oxaloacetate transamidase (200 U/mg, Roche)]. The NADP cycling indicator step (see Scheme IV) is begun by adding 1 ml of 6-phosphogluconate reagent [50 mM imidazole-HCl, pH 7.0, 25 mM acetic acid, 1 mM EDTA, 30 mM ammonium acetate, 5 mM MgC12, 0.1 mM NADP +, and 2.5 /zg/ml 6-phosphogluconate dehydrogenase (20 U/mg, Sigma)]. These 41s. s. Lin, J. K. Manchester,and J. 1. Gordon,J. Biol. Chem.276, 36000 (2001).

194

SPEC~LIZED USES

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Glutamateoxaloacetate Glutamate, . ~ . ~ s a m i ~ ot-Ketoglutarate Malate /~f Malate~ d ~ d r o g e ~ e ~ ¢ , Oxaloacetate NAD+ f f

~

Aspartate

NADH SCHEMEHI. Indicatorstep for NAD cycle.

6-Phosphogluconate 6-Phosphogluconate, , , ~ d r o g ~ Ribulose5-phosphate NADP+ ~

NADPH

SCHEMEIV. Indicator step for NADPcycle.

F-1,6bP

~

F-6P

Pi F-6P

Phosphoglucoisomerase

G-GP NADPH ",..~ 6edH H...~¥ If-

NADP+

6-PG

SCHEMEV. Schemefor fructose-l,6 bisphosphatase assay. indicator reactions are incubated for 10 min at 25 °. NADH generated from malate or NADPH produced from 6-phosphogluconate are measured fluorimetrically (excitation monitored at 365 nm, emission at 460 nm). Care is taken to ensure that for each enzyme, metabolite, or nucleotide determination, product formation is linear with respect to the range of cell extract used. Standards consisting of the metabolite of interest, or the product of the enzyme being assayed, are run in parallel with cell extracts. NAD + or NADP + standards are added at the cycling step in a minus extract control to coincide with levels produced by the experimental reactions. A known amount of malate or 6-phosphogluconate, corresponding to the predicted concentration of pyridine nucleotide produced from the cycling reaction, is included as a third reference control. See Ref. 41 for further details about calculating the amount of standards to include in these reactions.

Example: Fructose-l,6-bisphosphatase An assay for measuring fructose-1,6-bisphosphatase is presented in Scheme V as an example of how pyridine nucleotide-based enzyme cycling can be applied to

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LCM cell populations. Lysate (0.1 #1) is added to 0.5/zl of a solution containing 50 mM imidizole-HC1 (pH 7.0), 1 mM EDTA, 0.05% BSA, 2 mM MgC12, 0.2 mM NADP +, 0.2 mM fructose 1,6-bisphosphate, 0.25 U/ml yeast phosphoglucoisomerase (specific activity 350 U/mg; Roche), and 0.18 U/ml yeast glucose-6phosphate dehydrogenase (G6PdH). Also included at this step are 0.1-/zl aliquots of 5 and l0/zM fructose 6-phosphate which serve as internal standards. Following a 60 min incubation at 20 °, 0.5 #l of0.15 M N a O H is added, and the samples are incubated at 80 ° for 20 min. A 0.5-/zl aliquot is removed and added to 0.1 ml of NADP cycling reagent [100 mM imidizole-HC1 (pH 7.0), 7.5 mM t~-ketoglutarate, 5 mM glucose 6-phosphate (G-6P), 25 mM ammonium acetate, 0.02% BSA, 0.1 mM ADP, 1.5 U/ml glutamate dehydrogenase (GDH, specific activity = 120 U/mg), and 1.5 U/ml G6PdH] (see Scheme II). After a 60 min incubation at 38 °, samples are incubated for an additional 5 min at 100 °. Once cooled to 25 °, 1 ml of 6-phosphogluconate indicator reagent is added (see Scheme IV). The subsequent production of NADPH, corresponding to the amount of 6-PG produced in the cycling step, is determined fluorimetrically.

Preparation of Extracts for Assay of Cellular Metabolites The same number of cells is needed for analysis of metabolites as for enzymes. Different metabolites have different stabilities at different pH values. Therefore, an alkali as well as an acid extract is made from a single cap of captured cells. First, 1.2/zl of ice-cold 0.05 M NaOH containing 1 mM EDTA is added to a cap containing freeze-dried cells. Two 0.6-/zl aliquots are transferred to an "oil well." The first aliquot is designated as the alkali extract. The second aliquot is added to 0.6 #1 0.1 M HCI and is designated as the acid extract. Both extracts are heated to 80 ° for 20 min. The alkali extract is neutralized by adding 0.6 #1 of 100 mM Tris-HC1 (pH 8.1) and 0.05 M HC1. The acid extract is neutralized by adding 0.3/zl of 0.4M Tris-base. Extracts are stored at - 8 0 ° under vacuum until needed. The soluble protein content of the extract is determined using the colloidal gold method. A 0.2-/zl aliquot of the alkali extract is added to 0.5 ml colloidal gold reagent. Following a 60 min incubation at 37 °, absorbance is determined at 595 nm. Metabolite levels are determined using "oil wells" and the schemes described above with minor modifications. Acid or alkali extracts are employed depending on the metabolite. 41

Example: Glucose (Scheme VI) A 0.1-#1 aliquot from the acid extract is added to 0.1 #1 of a solution containing 50 mM Tris-HC1, pH 8.0, 0.04% BSA, 0.4 mM DTT, 0.1 mM NADP +, 4 mM MgC12, 0.5 m M ATP, 0.08 U/ml G6PdH, and 0.5 U/ml yeast hexokinase (specific activity 450 U/mg). The reaction mixture is incubated for 20 min at 20 ° followed by 0.1 /zl 0.1 M NaOH. The mixture is incubated for 20 min at 80 °. Five/zl of NADP cycling reagent (composition described above with the exception that GDH

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ATP~ HK~ADP Glucose

~'Jk'G-6P~6PdH .I#6-PG NADP+ ~

~

NADPH

SCHEME VI. Glucose assay.

and G6PdH concentrations are doubled) is added to each sample and the solution incubated for 60 rain at 38 °. Cycling is terminated with 0.5/zl 1 M NaOH and heating to 80 ° for 20 min. A 5-/zl aliquot is transferred to 1 ml 6-PG indicator reagent and NADPH fluorescence is determined.

[16] Laser Capture Microdissection in Pathology By FALKO FEND,

KATJA SPECHT, MARCUS KREMER,

and LETICIAQUINTANILLA-MARTfNEZ Introduction The molecular genetic analysis of pathologically altered tissues has greatly increased our understanding of the etiologies and pathogenesis of human disease processes. The identification of recurrent genetic alterations has a major impact on the pathologic diagnosis of cancer, and conventional morphological tumor classification will rapidly be replaced by defining disease entities based on the integration of clinical, morphological, phenotypical, and genetic information. Moreover, the establishment of individual molecular profiles of tumors may help to identify targets for specific therapeutic intervention. However, primary tissues are a complex mixture of various cell types, and tumors contain an abundance of reactive stromal and inflammatory cells, which frequently outnumber the neoplastic population. This inherent complexity can crucially influence the results of molecular genetic examinations of primary tissues, since many alterations such as loss of heterozygosity or point mutations in tumor suppressor genes or oncogenes can go undetected by standard detection methods if the percentage of "contaminating" stromal cells reaches a certain threshold. In expression profiling of bulk tissue, admixed cell populations can potentiaUy obscure tumor-specific signatures and can make message assignment to specific cell types impossible. Furthermore, early pathologic lesions, such as dysplasia or carcinoma in situ, are frequently inaccessible for conventional molecular analysis.


Copyright2002,ElsevierScience(USA). All rightsreserved. 0076-6879102 $35.00

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ATP~ HK~ADP Glucose

~'Jk'G-6P~6PdH .I#6-PG NADP+ ~

~

NADPH

SCHEME VI. Glucose assay.

and G6PdH concentrations are doubled) is added to each sample and the solution incubated for 60 rain at 38 °. Cycling is terminated with 0.5/zl 1 M NaOH and heating to 80 ° for 20 min. A 5-/zl aliquot is transferred to 1 ml 6-PG indicator reagent and NADPH fluorescence is determined.

[16] Laser Capture Microdissection in Pathology By FALKO FEND,

KATJA SPECHT, MARCUS KREMER,

and LETICIAQUINTANILLA-MARTfNEZ Introduction The molecular genetic analysis of pathologically altered tissues has greatly increased our understanding of the etiologies and pathogenesis of human disease processes. The identification of recurrent genetic alterations has a major impact on the pathologic diagnosis of cancer, and conventional morphological tumor classification will rapidly be replaced by defining disease entities based on the integration of clinical, morphological, phenotypical, and genetic information. Moreover, the establishment of individual molecular profiles of tumors may help to identify targets for specific therapeutic intervention. However, primary tissues are a complex mixture of various cell types, and tumors contain an abundance of reactive stromal and inflammatory cells, which frequently outnumber the neoplastic population. This inherent complexity can crucially influence the results of molecular genetic examinations of primary tissues, since many alterations such as loss of heterozygosity or point mutations in tumor suppressor genes or oncogenes can go undetected by standard detection methods if the percentage of "contaminating" stromal cells reaches a certain threshold. In expression profiling of bulk tissue, admixed cell populations can potentiaUy obscure tumor-specific signatures and can make message assignment to specific cell types impossible. Furthermore, early pathologic lesions, such as dysplasia or carcinoma in situ, are frequently inaccessible for conventional molecular analysis.


Copyright2002,ElsevierScience(USA). All rightsreserved. 0076-6879102 $35.00

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To circumvent these problems and to obtain more homogeneous cell populations for molecular analysis, manual and micromanipulator-based microdissection techniques have been developed during the past decade. 1-5 Despite the unquestionable progress achieved with these approaches, such as the unveiling of the lineage and clonality of the malignant cells in Hodgkin's disease, 6 the time-consuming nature of microdissection and the significant manual dexterity required for it have until recently prevented its broad application in pathology. The development of easy-to-handle, laser-assisted technologies such as laser capture microdissection (LCM) or laser microbeam microdissection (LMM) allows rapid and highly precise procurement of purified cell populations suitable for a variety of downstream analyses.7-11 For many diagnostic applications, the use of microdissected cells as template source requires few, if any, modifications of standard protocols, and LCM can easily be integrated into the routines of a molecular pathology laboratory. This chapter gives an overview of applications for laser-assisted microdissection in pathology, focused on, but not restricted to, LCM. We describe various protocols for tissue preparation, microdissection, and DNA and RNA analysis from microdissected tissues. Tissue Preparation Both fresh frozen tissues and fixed, paraffin-embedded specimens, as well as cytological preparations, can be used for LCM. The major difference between routinely fixed and frozen specimens lies in the amount and quality of nucleic acids and protein which can be isolated from these sources. However, pre-LCM tissue preparation and microdissection itself are also critically influenced by the type of starting material. 1 E d'Amore, J. A. Stribley, T. Ohno, G. Wu, R. S. Wickert, J. Delabie, S. H. Hinrichs, and W. C. Chan, Lab. Invest. 76, 219 (1997). 2 G. Deng, Y. Lu, G. Zlotnikov, A. D. Thor, and H. S. Smith, Science 274, 2057 (1996). 3 R. Kiippers, M. Zhao, M. L. Hansmann, and K. Rajewsky, EMBO J. 12, 4955 (1993). 4 L. Whetsell, G. Maw, N. Nadon, E D. Ringer, and E V. Schaefer, Oncogene 7, 2355 (1992). 5 j. j. Going and R. E Lamb, J. Pathol. 179, 121 (1996). 6 R. Kiippers, K. Rajewsky, M. Zhao, G. Simons, R. Laumann, R. Fischer, and M. L. Hansmann, Proc. Natl. Acad. Sci. U.S.A. 91, 10962 (1994). 7 M. BShm, I. Wieland, K. Schiitze, and H. Rithben, Am. J. Pathol. 151, 63 (1997). 8 R. E Bonner, M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997). 9 M. R. Emmert-Buck, R. E Bonnet, E D. Smith, R. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 10 L. Fink, W. Seeger, L. Ermert, J. Hanze, U. Stahl, E Grimminger, W. Kummer, and R. M. Bohle, Nat. Med. 4, 1329 (1998). II K. Schtitze and G. Lahr, Nat. Biotechnol. 16, 737 (1998).

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Paraffin-Embedded Tissues Paraffin embedding, usually preceded by fixation in neutral, buffered 10% formalin, is still the most widely used method for the processing and conservation of diagnostic pathological specimens. However, cross-linking fixatives, such as formalin, lead to significant fragmentation of nucleic acids and cannot be used for techniques such as Southern or Northern blot analysis, which require large amounts of high molecular weight DNA and RNA, respectively. Nevertheless, microdissected paraffin-embedded tissues can serve as template sources for PCR-based analyses of both DNA and RNA, as long as relatively small amplicon sizes are used. Several groups have made efforts to replace formalin with precipitating fixatives such as ethanol, followed by paraffin embedding for improved preservation of nucleic acids.12' 13 Although this represents a promising step to ensure both excellent morphology and superior quality of biologic macromolecules, formalin-fixed specimens still represent the biggest source of archival material for molecular studies. The preparation of paraffin sections for LCM and subsequent DNA or RNA extraction requires little deviation from standard laboratory procedures. Paraffin sections are cut under precautions against cross contamination between different tissue samples, then mounted on standard or plus-charged glass slides, depending on the subsequent staining procedure. After drying at 60 °, usually overnight, the slides are dewaxed in xylene 2x for 5 min, rehydrated through graded alcohols, and finally immersed in distilled water. If the slides are used for RNA extraction, nuclease-free water (DEPC-treated water) should be employed. Hematoxylin-Eosin Staining. The slides are stained in Mayer' hematoxylin solution (Sigma-Aldrich, Deisenhofen, Germany) for 30 sec to 1 min, followed by blueing solution, 70% ethanol, eosin (5-20 sec), and dehydration through graded alcohols and 2 changes of xylene. The use of fresh, 100% ethanol as the final step before xylene is of great importance, because residual humidity can severely interfere with tissue transfer during LCM. Stained, dehydrated slides should be used for LCM as soon as possible, because prolonged storage may lead to reduced tissue transfer and may also influence the quality of nucleic acids. Stained slides should be stored in the presence of desiccants. Several other staining techniques such as hematoxylin or hemalum only, nuclear fast red, and others have been tested and may give equivalent or superior morphology, depending on the type of tissue. 14 Immunohistochemical Staining for Paraffin Sections. A drawback of laserassisted microdissection is the requirement of dehydrated tissue sections or cell 12 S. M. Goldsworthy, E S. Stockton, C. S. Trempus, J. E Foley, and R. R. Maronpot, Mol. Carcinog. 25, 86 (1999). 13 M. Shibutani, C. Uneyama, K. Miyazaki, K. Toyoda, and M. Hirose, Lab. Invest. 80, 199 (2000). 14 T. Ehrig, S. A. Abdulkadir, S. M. Dintzis, J. Milbrandt, and M. A. Watson, J. Mol. Diagn. 3, 22 (2001).

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preparations without coverslip, which leads to a significant decrease in optical resolution and loss of cytological detail. Although routinely stained sections (e.g., hematoxylin-eosin) are frequently sufficient for target recognition in wellstructured tissues, precise microdissection from tissues lacking easily identifiable architectural features such as lymphoid tissues, inflammatory infiltrates, or diffusely infiltrating neoplasms can be virtually impossible. In practice, the precision of laser-assisted microdissection is more frequently limited by difficulties in recognizing the target cells rather than by the technical specifications of the dissection tool. Immunohistochemical or cytochemical staining techniques can improve precision of LCM by rendering high-contrast targets and further allow separation of morphologically homogeneous cell populations according to phenotypical or functional criteria. Standard immunohistochemical procedures for paraffin-embedded tissues do not interfere with LCM and do not seem to have a major influence on subsequent DNA recovery. 1'15 Our laboratory uses for most part routine staining protocols for diagnostic immunohistochemistry, including appropriate heat-induced antigen retrieval, as determined by the primary antibody used. To prevent detachment of slides during the staining procedure, plus-charged (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) or coated (e.g., with poly-L-lysine) slides should be used, which do not interfere with tissue transfer during LCM if handled properly. For the detection of most primary antibodies, the ABC (avidin-biotin complex) technique is employed, with a biotinylated secondary antibody and horseradish peroxidase-labeled avidin as third step. Diaminobenzidine (DAB) as chromogen is well suited for LCM, since the stained sections can be counterstained with hemalum and dehydrated as above, and the stain does not interfere with PCR. In fact, the color precipitate remains on the membrane after digestion or elution of captured cells and can serve as visual control and documentation of dissection specificity (Fig. l). Since strong staining results may be beneficial for the identification of the targeted cells, standard protocols may be optimized accordingly, including overnight incubation with the primary antibody or increased antibody concentrations. A short immunostaining protocol for frozen sections designed to reduce the exposure to aqueous media is described below. LCM of Paraffin Sections. LCM of paraffin sections, whether routinely stained or immunostained, is usually straightforward, and even archival, stained sections can be used successfully after removal of the coverslip with xylene. However, some problems may be encountered during LCM. 1. Poor visualization of targeted cell population. Depending on fissue architecture and type of staining, poor visualization can severely compromise dissection

15E Fend,L. Quintanilla-Martinez,S. Kumar,M. W. Beaty,L. Blum,L. Sorbara, E. S. Jaffe,and M, Raffeld,Am. J. Pathol. 154, 1857(1999).

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A

B

.

,

FIG. 1. (A) LCM of a paraffin section of a composite non-Hodgkin's lymphoma immunostained for CD5. The neoplastic follicles are CD5 negative (asterisks), whereas the interfollicular neoplastic cells express CD5, in addition to reactive T-cells. The holes left behind by the procedure are clearly visible. (Three-step immunoperoxidase technique, x100.) Amplification of rearranged immunoglobulin heavy chain genes from the microdissected CD5+ and C D 5 - cell populations repeatedly rendered two products of different size and sequence (not shown), confirming the presence of two different clones. IF. Fend, L. Quintanilla-Martinez, S. Kumar, M. W. Beaty, L. Blum, L. Sorbara, E. S. Jaffe, and M. Raffeld, Am. J. Pathol. 154, 1857 (1999)]. (B) Cap surface after proteinase K digestion. Although the cellular material has already been removed, the outlines of the captured cells immunostained for CD5 are clearly visible due to the residual DAB precipitate which remains on the thermoplastic membrane and allows control of dissection specificity.

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specificity. Here are some remedies: Use the diffuser or add a drop of xylene to the slide, which works like a coverslip, and create a roadmap image. After evaporation of the xylene, use this image as guidance. Reduce the time in hematoxylin or change to another stain. For certain tissues, the use of immunostaining may be mandatory. Mounted and stained parallel sections can also aid to "navigate" on the slide used for LCM, but this is only appropriate for tissues with larger, predictable anatomical structures which can be followed through step sections. 16 2. The captured tissue remains on the slide. This is probably the most vexing problem. Frequently, insufficient dehydration is the reason, and reimmersion of the section in absolute ethanol followed by xylene may be a remedy. Another strategy is to incubate the slide in water with 3% glycerol before dehydration, which can be of help for various cell or tissue preparations. 17 Uneven section surfaces, slightly tilted placement of the cap, or repositioning of the cap after dissection with adherent cells on the lower surface can compromise tissue contact and dissection efficiency. DNA Extraction. After control of dissection specificity and, if necessary, removal of nonspecifically adherent cells with a light adhesive, put the cap on a 500-/zl tube which contains 50-100/zl of TE buffer with 400/zg/ml proteinase K. The optimal buffer volume depends on the amount of captured cells. For small numbers of cells, the area containing the captured cells can be cut from the cap surface under the microscope with a sterile blade and directly immersed into 10-20 #1 of proteinase K-containing buffer. Altematively, specially designed caps for small amounts of cells can be used in conjunction with the micro-extraction chamber suitable for small fluid volumes (Arcturus Engineering, Santa Clara, CA). Unless a membrane fragment is directly immersed in the buffer, the tube carrying the cap is inverted and incubated at 55 ° for 4 - 8 hr or overnight. Despite the small amount of cells, complete digestion of cells from paraffin-embedded sources requires at least several hours. It is advisable to control for complete digestion by restaining the cap to detect any remaining cell fragments. Because of the limited amount of cells, proteinase K digestion without subsequent purification steps (e.g., organic extraction) is usually sufficient for standard PCR. After heat inactivation of proteinase K and spinning down of any particulate debris, the supernatant can be used for PCR directly. Determination of B-Cell Clonality by PCR Using Consensus Primers against Framework Three Region (FR3) of Immunoglobulin Heavy Chain Genes (IgH). The determination of B- or T-cell clonality of lymphoid proliferations is one of the 16M. H. Wong,J. R. Saam, T. S. Stappenbeck,C. H. Rexer, and J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 97, 12601 (2001). 17L. Jin, C. A. Thompson,X. Qian, S. J. Kuecker,E. Kulig, and R. V. Lloyd, Lab. Invest. 79, 511 (1999).

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most frequently used diagnostic molecular assays in pathology. Since the percentage of clonal cells must reach a certain threshold of at least 2 - 3 % - - i n reality often closer to 10% of the total cell population--to be reliably detected, microdissection can be used to enrich the cell population in question. Besides Hodgkin's disease, many other lesions such as nodular lymphoid infiltrates in the bone marrow or extranodal locations or composite lymphoma lend themselves to microdissection (Fig. 1). However, the use of microdissected tissue fragments for clonality determination requires rigorous control and careful interpretation of results, since the small amounts of lymphoid cells serving as template for amplification can result in "pseudoclonal" amplification products, which should not be equated with malignancy. The effects of formalin fixation with decrease in template quality and subsequent preferential amplification of some rearrangements over others, due to the use of consensus primers with varying binding affinity, can further aggravate this problem. Whenever possible, a single-step PCR should be used to reduce the possibility of clonal amplification products of uncertain relevance. If larger amounts (hundreds to thousands) of cells can be collected, conventional single-step PCR is sufficient to generate enough PCR product for fragment length analysis, direct sequencing, or cloning. Perform all reactions in duplicate, preferably using cells from different microdissections. If clonal bands are not identical in all reactions, they are likely the result of preferential amplification of rare B cells. The following protocol is currently used in our laboratory 15'18: The reaction volume of 25 /zl contains 0.4/zM/liter of each primer (FR3a and LJH19), 0.2 mM/liter dNTPs, 2 mM/liter MgC12, 1.25 U of Taq polymerase (Amplitaq Gold, PerkinElmer, Weiterstadt, Germany) and 1-5 /zl of template DNA. After initial denaturation at 94 ° for 4 min, 40 cycles of amplification are performed at 94 ° for 1 min, 56 ° for 30 sec, and 72 ° for 30 sec, followed by a final extension step at 72 ° for 10 min. The PCR products can be run on a 3% Metaphor gel (FMC Bioproducts, Rockland, ME) or on a 16% polyacrylamide gel. For computer-assisted fragment length analysis, one of the primers is end-labeled with fluorescein, and amplification is performed under identical conditions. Fluorescein-labeled PCR products are analyzed on a high-resolution polyacrylamide gel using an ABI Prism 377 automated sequencer and Genescan software (PerkinElmer). If only very small numbers of cells are available, such as groups of Hodgkin and Reed-Sternberg cells, a seminested protocol using an internal primer directed against homologous sequences of the IgH joining region genes (VLJH) is necessary. 2° After first-round amplification as described above, another 25 cycles under identical conditions are performed, replacing the LJH primer with the VLJH 18M. Kremer,A. D. Cabras,E Fend, S. Schulz, K. Schwarz,H. Hoefler,and M. Werner,Hum. Pathol. 31, 847 (2000). 19G. H. Segal,T. Jorgensen.A. S. Masih, and R. C. Braylan,Hum. Pathol. 25, 1269 (1994). 20M. Kremer,M. Sandherr,B. Geist,A. D. Cabras,H. HSfler,and E Fend,Mod. Pathol. 14, 91 (2001).

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primer and using 3-5 #1 of the first-round product, both undiluted and 1 : 10 diluted, as template for the second round. The isolation of groups of 30 to 50 RS cells per cap and their joint analysis greatly reduce the work needed for clonality analysis of single cells and allow repeat amplification or investigation of other genes. Since contamination by occasional nonneoplastic lymphocytes is likely with this approach, cloning and sequencing of PCR products obtained from several distinct groups of RS cells originating from different microdissections is necessary to confirm the tumor cell origin of the amplification product. Amplified bands are purified from agarose or polyacrylamide gels with appropriate techniques, ligated into the PCR2.1 vector (TA cloning kit, Invitrogen, Carlsbad, CA), and cloned into INVaF' bacteria. 15,2° Sequencing of several inserts obtained from each PCR product will confirm or disprove the clonal identity of the isolated cells, and a minority of contaminating clones will not interfere with the interpretation. RNA Extraction and Real-Time TaqMan RT-PCR in Formalin-Fixed, ParaffinEmbedded Tissues. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues is generally of poor quality because degradation of the RNA can occur before completion of the formalin fixation process. Moreover, as mentioned above, formalin causes cross-linkage of nucleic acids and proteins and covalently modifies RNA by the addition of monomethylol groups to the bases, making RNA extraction, cDNA synthesis, and quantitation analysis problematic. 21 When performing gene expression analysis in FFPE tissues, it is therefore extremely important (a) to choose an RNA extraction procedure that provides only minimally cross-linked RNA and (b) to select very small target sequences in a range of 60-100 bp for real-time RT-PCR, enabling the detection of fragmented and deg r a d e d R N A . 22,23 Generally, there is a huge number of methods available for RNA extraction; however, in our experience, the most successful method in terms of yield of extractable RNA and suitability of the RNA for real-time RT-PCR analysis involves proteinase K digestion. 22 Briefly, RNA from a small number of microdissected cells (20-10,000 cells) is extracted using a modification of the method described by Rupp and L o c k e r . 24 Microdissected cells are transferred to a 1.5-ml microcentrifuge tube and lysed in 200/zl lysis buffer, containing l0 mmol/liter Tris-HC1 (pH 8.0), 0.1 mmol/L EDTA (pH 8.0), 2% sodium dodecyl sulfate (pH 7.3), and freshly added 500 #g/ml proteinase K. Cells are digested for 12 hr at 60 ° until the tissue is completely solubilized. After heat inactivation of the proteinase K for 5 min at 95 °, the RNA is purified by phenol/chloroform extraction: 1/10 volumes 2 M sodium acetate (pH 4.0 ), 1 volume water-saturated acidic 21 N. Masuda, T. Ohnishi, S. Kawamoto, M. Monden, and K. Okubo, Nucleic Acids Res. 27, 4436 (1999). 22 K. Specht, T. Richter, U. MUller, A. Walch, M. Werner, and H. H6fler, Am. J. Pathol. 158, 419 (2001). 23 A. E. Krafft, B. W. Duncan, K. E. Bijwaard, J. K. Taubenberger, and J. H. Lichy, Mol. Diagn. 2, 217 (1997). 24 G. M. Rupp and J. Locker, BioTechniques 6, 56 (1988).

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phenol, and 1/5 volume chloroform are added to the reaction. After vortexing, the samples are put on ice for 15 min and then centrifuged at 14,000 rpm for 20 rain at 4 °. The upper, aqueous phase containing the RNA is transferred to a new microcentrifuge tube and the RNA is precipitated with 2/zl of 10 mg/ml carrier glycogen and 1 volume isopropanol. After incubation for 2 hr at - 2 0 °, the RNA is pelleted by centrifugation at 14,000 rpm for 20 min. The pellet is washed once with 70% ethanol, dried, and resuspended in 20/zl RNase-free H20. If the primers and probes used for subsequent real-time RT-PCR do not span an intron, the removal of genomic DNA by DNase digestion is necessary at this point. cDNA synthesis reaction is carried out with Superscript II Reverse Transcriptase in a final reaction volume of 20/zl as described in the instruction manual (Superscript Choice system) provided by Life Technologies. One-half of the isolated RNA (10/zl) is used for reverse transcription, while a no RT control reaction should be performed in parallel with the other half of the RNA. RNA is annealed with 250 ng random primers at 25 ° for 10 min and then reverse transcribed with 200 U (1 /zl) Superscript reverse transcriptase in 4 #1 of 5× first-strand buffer [250 mM Tris-HCL (pH 8.3), 375 mM KCL, 15 mM MgCI2], 10 mM dithiothreitol (DTT), 1/zl of 0.5 mM of each dNTP, and 1 /zl of RNase inhibitor (40 U)] for 60 min at 42 °. Typically, 1/10-1/20 of the cDNA reaction is then used for subsequent real-time TaqMan PCR. Frozen Tissues

Although frozen tissue represents a superior source for intact biomolecules compared to FFPE tissues, the morphology of frozen sections frequently is poor and further compromises the precision of microdissection. As mentioned above, IHC is an excellent tool for improving the visualization of the target populations. However, standard IHC protocols require several hours of incubation in aqueous media, which results in a significant loss of RNA through the action of ubiquitous RNases. Therefore, several groups have developed immunostaining protocols suitable for LCM and subsequent gene expression analysis. 17,25,26The following rapid immunostalning protocol can be performed with many different primary antibodies and standard reagents and renders mRNA of good quality, although a loss of mRNA in comparison to rapid routine staining (e.g., hematoxylin-eosin) does still occurY Immuno-LCM o f Frozen Sections. The Quick Staining kit (DAKO Corp., Carpinteria, CA) used in the initial publication is no longer commercially avallableY Alternatively, the procedure can be performed with analogous reagents suitable for quick immunostaining, such as a three-step (strept-)avidin-biotin 25E Fend,M. R. Emmert-Buck,R. Chuaqui,K. Cole,J. Lee,L. A. Liotta,andM. Raffeld,Am. J. Pathol. 154, 61 (1999). 26H. Murakami,L. Liotta,and R, A. Star, Kidney Int. 58, 1346(2000).

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FIG. 2. LCM of a frozen section of Hodgkin's disease immunostained for the CD30 antigen. The holes left behind are clearly visible, as well as the membrane staining of other tumor cells. The arrow denotes granulocytes, which show positivity due to endogenous peroxidase activity. (Rapid three-step immunoperoxidase technique, x 600.)

system with a biotinylated secondary antibody and horseradish peroxidase (HRP) (strept-)avidin complex optimized for high sensitivity (Fig. 2). A useful method is the employment of secondary antibodies coupled to a polymer backbone carrying multiple HRP molecules (EnVision, DAKO), which abolish the necessity for a third incubation step before color development and show increased sensitivity. Furthermore, the system is biotin-free and therefore lacks background staining as a result of endogenous biotin. 27

Staining procedure Frozen sections are mounted on charged slides (Superfrost Plus) and immediately refrozen on dry ice. Sections can be stored at - 8 0 °. The sections are briefly thawed at room temperature and immediately immersed in cold acetone for 1-2 min. Drying of the sections before fixation will severely compromise subsequent tissue capture. Do not stain more than 1-3 sections at one time, because multiple slides will lead to a significantly prolonged incubation time. After evaporation of acetone, the slides are rinsed briefly in buffered PBS or Tfis pH 7.4 and incubated with 70-100/zl primary antibody for 1.5-3 rain. 27 U. K'mmnerer, M. Kapp, A. M. Gassel, T. Richter, C. Tank, J. Dietl, and E Ruck, J. Histochem. Cytochem. 49, 623 (2001).

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The optimal dilution and staining time has to be determined individually. The slide is rinsed briefly with buffer and incubated with the secondary reagent for 2-3 min. If a three-step avidin-biotin system with a biotinylated secondary antibody is used, it is followed by incubation with the streptavidin-HRP complex for 2-3 min. Subsequently, the slide is covered with 100 /zl of freshly prepared 3,3'diaminobenzidine solution for 2-5 min. Then the slide is rinsed in water and briefly counterstained with hematoxylin (20-30 sec) if desired. The sections are dehydrated through graded alcohols (15-30 sec each, including twice 100% ethanol) and xylene (twice for 2 rain each). For all aqueous solutions, the use of pure, RNase treated water is recommended. During the incubation steps, placental RNase inhibitor (PerkinElmer) may be added in a concentration of 200-400 U/ml. RNA Extraction. After LCM, the tubes carying the caps with the captured cells are put on ice. RNA extraction is performed using lysis in guanidinium isothiocyanate followed by extraction with water-saturated phenol and subsequent precipitation with cold isopropanol and glycogen added as carrier (Micro RNA isolation kit, Stratagene, La Jolla, CA). If the presence of contaminating DNA is critical, DNase digestion is mandatory. RNA is dissolved in pure water containing 1 /zl of RNase inhibitor and incubated for 2 hr at 37 ° with 10-20 U of DNase I (Genhunter Corp., Nashville, TN). After reextraction of RNA following the same protocol as above, the RNA is dissolved in pure water, and 1/xl of RNase inhibitor is added. Reverse transcription is performed with 2.5/zmol/liter of random hexamers, 250/zmol/liter of each dNTP, and 100 U of MMLV reverse transcriptase (GenHunter) in a final volume of 20/zl. A mock reaction without RT should be performed in parallel.9,15 Depending on the amount of isolated cells, 20/zl of cDNA is usually sufficient for 10-20 or more single-step PCR reactions, and fragments larger than 500 bp can be amplified successfully. Conclusions The above protocols represent a small selection of fairly simple, easy-to-use techniques which can be readily performed in most molecular pathology laboratories. However, methodical improvements and further technical developments are realized at a rapid pace in all relevant areas, including tissue conservation and pre-LCM preparation, identification, and precise isolation of targeted cells, as well as more sensitive downstream analytical techniques adapted to small amounts of tissue, including high throughput screening methods such as cDNA microarrays or proteomics. Our increasing ability to correlate molecular findings with morphology and phenotype on the microscopic level will have a profound impact on all aspects of pathology.

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[17] Use of Laser Capture Microscopy in the Analysis of Mouse Models of H u m a n Diseases By MERAL J. ARIN and DENNIS R. ROOP Introduction The use of laser capture microdissection (LCM) has been a major step toward a better understanding of the molecular mechanisms that play a role in various disease processes. 1 The ability to readily obtain and isolate cells of interest from complex tissues has made LCM an attractive technology in the fields of genomics and proteomics. To study genetic changes that occur in a particular cell type, tiny subpopulations of cells need to be isolated to reduce the risk of contamination. Before the development of LCM, this process of isolating specific cells of interest was very tedious, irreproducible, and inefficient.2 The skin is an attractive organ for the application of LCM since it is composed of a complex mixture of different cell types. Keratinocytes represent the major cell type in the epidermis, the outer layer of the skin. They contain highly polymeric molecules, the keratins, that confer mechanical stability on these cells. Beneath the epidermis lies the dermis which is an elastic support structure and contains fibroblasts as the predominant cell type. Dermal fibroblasts synthesize the structural components of the dermis which include collagen and elastic fibers as well as ground substance. Molecular defects in structural components of the epidermis and dermis have been identified in several hereditary blistering disorders of the skin. 3 These disorders are characterized by mutations in various genes encoding intra- and extracellular molecules leading to blister formation in distinct layers of the skin. We describe the use of LCM in the analysis of animal models of hereditary blistering disorders. Animal Models of Hereditary Blistering Disorders Animal models are valuable tools to study pathways involved in disease processes. Several animal models have been generated and are currently being used to study the molecular and cellular basis of genetic skin disorders. 4 For the analysis of two hereditary blistering disorders, epidermolytic hyperkeratosis (EHK) and 1 M. R. Emmert-Buck, R. E Bonner, E D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 2 j. j. Going and R. E Lamb, J. Pathol. 179~ 121 (1996). 3 B. P. Korge and T. Krieg, J. Mol. Med. 74, 59 (1996). 4 M. J. Arin and D. R. Roop, Trends Mol. Med. 7, 422 (2001).


Copyright2002,ElsevierScience(USA). All rightsreserved. 0076-6879,'02 $35.00

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epidermolysis bullosa simplex (EBS), we generated mouse models that reproduce the human disease at both the phenotypic and genetic levels. To understand the role of stem cells in these disorders, we generated mouse models that allow focal induction of the EBS and EHK phenotypes in a circumscribed area of the skin. 5'6 In these mouse models, we used LCM to isolate epidermal keratinocytes from stained tissue sections obtained from areas of interest (see below). Tissue Microdissection Several methods have been developed to isolate subpopulations of cells from tissue sections. These include ablation of unwanted regions and subsequent collection of the remaining cells and manual separation of the ceils of interest by fine needle, pipette, or blade, as well as irradiation of manually ink-stained sections to destroy unwanted genetic material. 7's These conventional techniques are very time consuming and require a high degree of manual dexterity, which limits their practical use. Compared to LCM, which uses a laser pulse to precisely target the cells of interest, these techniques have proved ineffective and nonefficient. Using LCM, it has been possible to reliably isolate pure populations of cells from tissue sections under microscopic visualization. To perform LCM, a histological section containing the tissue of interest is placed under the specifically designed microscope and the image is transferred to a computer screen. A transparent thermoplastic membrane is mounted on an optically clear cap which fits on a standard 0.5-ml microcentrifuge tube for further processing. The cap is positioned to cover the area of interest. Cells can then be melted onto the ethylene vinyl acetate (EVA) membrane by a near infrared laser beam, which builds a strong focal bond between the cells and the film.1 The strong adherence of the tissue to the activated membrane allows selective removal of the cells of interest. Large numbers of cells can be isolated in a short time by repeating multiple laser impulses across the whole cap surface. The size of the laser beam spot can be selected at 7.5, 15, and 30/zm. We used a spot size of 30 # m in our experiments to capture 1-2 keratinocytes per laser pulse. The cells of interest can then be easily pulled away from the slide by simply lifting the membrane from the tissue slide. The cells are then ready for molecular analysis. This technique can be used on formalin-fixed, paraffin-embedded tissue sections, as well as on frozen tissues. The quantity of products amplified by PCR (see below) is higher from frozen tissue than from paraffin-embedded tissue. 9 5 M. J. Arin, M. A. Longley, X. J. Wang, and D. R. Roop, J. Cell. Biol. 152, 645 (2001). 6 T. Cao, M. A. Longley, X. J. Wang, and D. R. Roop, J. Cell. Biol. 152, 651 (2001). 7 L. Whetsell, G. Maw, N. Nadon, D. E Ringer, and E V. Schaefer, Oncogene 7, 2355 (1992). 8 D. Shibata, D. Hawes, Z. H. Li, A. M. Hernandez, C. H. Spruck, and E W. Nichols, Am. J. Pathol. 141, 539 (1992). 9 S. M. Goldsworthy, P. S. Stockton, C. S. Trempus, J. E Foley, and R. R. Maronpot, Mol. Carcinog. 25, 86 (1999).

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The most important advantage of LCM is its speed combined with its precision. Thousands of cells can be captured within a few minutes. The exact morphology of microdissected cells is retained during this procedure and can be visualized directly after microdissection. This allows control and documentation of captured cells throughout the procedure. Some limitations exist, however, including the considerable amount of time required when a large number of tissue samples is involved. In addition, the optical resolution obtained is limited because of scattering of transmitted light that passes through the air spaces within dehydrated tissue (and absence of a coverslip), which may limit the identification of cells of interest.l°, 11 In certain tissues, it is difficult to identify different cell types by morphology alone. Immuno-LCM allows a rapid and precise isolation of specific subpopulations of cells that express distinct proteins. The advantages of immunohistochemically stained frozen sections are good optical resolution and faster and more precise identification of stained cell populations. In addition, frozen sections have been shown to yield a much higher quality of RNA for various applications such as generation of expression libraries and screening of cDNA arrays.12 We used LCM to analyze keratinocytes from our disease models. In these models, topical application of RU486 to a focal area of the skin results in activation of the mutant keratin allele giving rise to focal areas of affected skin (see below). Keratinocytes from affected and adjacent nonaffected skin were used for LCM analysis. LCM in A n a l y s i s of M o u s e M o d e l s for M o s a i c S k i n D i s o r d e r s Mosaic skin disorders are characterized by the presence of at least two genetically distinct cell populations from the same differentiation lineage. The molecular and cellular mechanisms that lead to clinical mosaicism are poorly understood. It remains unclear why mosaicism exists for certain disorders and not for others. Two candidate disorders to analyze the mechanisms that lead to clinical mosaicism are the keratin disorders, epidermolytic hyperkeratosis (EHK) and epidermolysis bullosa simplex (EBS). EHK is caused by point mutations in the genes encoding keratins l and 10, leading to blister formation in the suprabasal layers of the epidermis. In EBS, blistering takes place in the basal compartment and is caused by point mutations in keratins 5 and 14. Whereas mosaic forms exist for EHK with linear lesions of affected and unaffected skin, this has never been described for EBSJ 3 To investigate the underlying cause of this phenomenon, we generated mouse models for both disorders. In these 10 S. Curran, J. A. McKay, H. L. McLeod, and G. I. Murray, MoL Pathol. 53, 64 (2000). ll E Fend and M. Raffeld, J. Clin. Pathol. 53, 666 (2000). 12 E Fend, M. R. Emmert-Buck, R. Chuaqui, K. Cole, J. Lee, L. A. Liotta, and M. Raffeld, Am. J. Pathol. 154, 61 (1999). 13 A. S. Paller, A. J. Syder, Y. M. Chan, Q. C. Yu, E. Hutton, G. Tadini, and E. Fuchs, N. Engl. J. Med. 331, 1408 (1994).

210

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SPECIALIZEDUSES CrePR1

K,,Promo,e, I C+

[

PR antagonist Cre transgene

wt allele

~ IoxP

mutK(P l~ IoxP

wt allele mutKlOIoxp

LCM

IoxP

FIG. 1. Schematic representation of LCM analysis of keratinocytes that were captured from the epidermis of an inducible mouse model that allows focal induction of the phenotype in a circumscribed area of the skin. In the example shown, bigenic mice were generated that contain a keratin 10-point mutation (*), a neomycin resistance gene cassette (neo) flanked by loxP sites on one allele (mutK10ne°), and an inducible Cre transgene (CrePR 1) under the transcriptional control of the keratin 14 promoter. These mice lack a phenotype due to suppression of the mutant allele by the presence of neo cassette in intron 1. Topical application of the inducer (RU486) to the skin of these mice results in excision of the neo cassette and restoration of expression of the mutant allele (mutK10 l°xP) with the expected phenotype at the site of induction. Skin biopsies were taken from previously treated, phenotypic areas and keratinocytes were isolated from the epidermis by LCM for PCR analysis to confirm that the neo cassette was excised in lesional areas.

mouse models expression of mutated keratin proteins can be focally induced in restricted areas of the skin using the ligand-inducible CrePR1 system. 14 Topical application of RU486 on these mice results in activation of CrePR1 (an inducible form of Cre recombinase, which recognizes very specific sequences termed loxP sites). In its activated form, CrePR1 excises the neomycin (neo) cassette, which is flanked by loxP sites and inhibits expression of the mutant keratin allele when inserted into the first intron. Excision of the neo cassette activates expression of the mutant keratin allele, resulting in a phenotype characteristic of either EHK or EBS (Fig. 1). We used LCM to capture keratinocytes from the epidermis from treated 14 C. Kellendonk, E Tronche, A. E Monaghan, E O. Angrand, E Stewart, and G. Schtitz, Nucleic Acids Res. 24, 1404 (1996).

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211

D

mtK10neo

mtK10IOxP

~ R156C

neo I~P__~ r'-"l

~/L K14.CREPR1+ RU486 IoxP ~ - ~

FIG. 2. Induction and characterization of blisters in K14-CrePR1/mtK10 he° mice. (A) RU486 was applied once a day for 3 to 5 consecutive days to the upper trunk and paws of newborn bigenic pups (+/mutK10ne°.CrePR1). A 6-day-old pup is shown, where blisters formed at the site of induction. After rupture of the blisters, scaling developed around the site of the previous blister. (B) Paw of the same mouse 5 months after induction shows the persisting phenotype with thick hyperkeratoses, as seen in older EHK patients. (C) H&E staining of a biopsy taken from a blistered paw shown in (A). The areas of cytolysis in the suprabasal layers of this section were subjected to LCM. (D) Schematic representation of the Cre-mediated excision of the neo cassette in skin areas treated with the inducer. [Modification of Fig. 2 in M. J. Arin, M. A. Longley, X. J. Wang, and D. R. Roop, J. Cell. Biol. 152, 645 (2001) by copyright permission of The Rockefeller University Press,]

a n d u n t r e a t e d areas. B i o p s i e s w e r e p r o c e s s e d as d e s c r i b e d b e l o w a n d s e c t i o n s were visualized using the PixCell LCM system (Arcturus Engineering, Mountain View, C A ) . D N A w a s e x t r a c t e d a n d s u b j e c t e d to P C R a n a l y s i s to d e t e r m i n e t h e p r e s e n c e or a b s e n c e o f the n e o cassette. I n the E H K m o d e l , w e f o u n d p e r m a n e n t e x c i s i o n o f t h e n e o cassette, a n d t h u s a c t i v a t i o n o f t h e m u t a n t allele, c o n f i r m i n g t h a t C r e - m e d i a t e d r e c o m b i n a t i o n o c c u r r e d i n e p i d e r m a l s t e m cells (Fig. 2). 5 O u r results i n d i c a t e t h a t in E H K , m u t a n t a n d w i l d - t y p e s t e m cells c a n c o e x i s t in t h e

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basal compartment, leading to a persistent phenotype. 5 In the EBS model, blisters formed after activation of the mutant allele; however, they healed after 10 days and never reappeared. We used LCM to document that the neo cassette was initially excised in the blistered area, but cells in these lesions were replaced by phenotypically normal epidermal stern cells (i.e., those surrounding the treated area that retained the neo cassette) (Fig. 3). 6 The schematic shown in Fig. 4 summarizes the results obtained with our inducible mouse models. Selective pressure against stem cells containing an activated EBS allele occurs because these stem cells express the mutant K14 protein. In the EHK model, the EHK allele is also activated in stem cells; however, it is the differentiated progeny of these ceils, not the stem cells themselves, that express the mutant K10 protein. These observations could explain why mosaic forms have been reported for EHK, but not EBS. P r o t o c o l for LCM in O u r A n a l y s i s o f D i s e a s e M o d e l s I. Skin biopsies are fixed in 10% formalin for a few hours to overnight; 2.5 to 4 hr seem to work best since DNA yield decreases with prolonged fixation times. After dehydration through graded ethanol, the sections are embedded in paraffin. 2. Tissue sections are mounted on plain, untreated, and uncharged glass slides. The use of charged slides increases the strength of adhesion of the section to the slide, which interferes with tissue capture. We routinely use 5-#m sections of paraffin-embedded tissue for capturing keratinocytes, but sections of between 5 and 20 ]zm in thickness can be used depending on the diameter of the nucleus of the cell of interest. Air dry sections overnight at room temperature. If sections do not adhere to the slide sufficiently they can be baked at 42 ° for up to 8 hr. 3. Deparaffinize 5-#m sections in xylene two times for 5 rain and rinse in a graded alcohol series (100%, 95%, 70% ethanol for 30 sec each) with a final rinse in distilled water for 30 sec. The slides are now ready for staining, e,g., with Nuclear Fast Red (Vector Laboratories, Burlingame, CA). 4. Fix the sections in 75% ethanol for 30 sec or in acetone for 4 min at 4 °. Transfer to distilled water for 30 sec. Stain with Nuclear Fast Red for 30-60 sec. Rinse in distilled water. Dehydrate 70% ethanol for 30 sec, 95% ethanol for 30 sec, I00% ethanol for 30 sec. Dip in xylene two times for 5 n-tin. Air dry for at least 20 rain in a hood. One reference slide is stained with H&E for identification of areas and cells of interest. 5. Sections are then visualized using the PixCell LCM system (Arcturus Engineering, Mountain View, CA). A thermoplastic polymer coating (ethylene vinyl acetate, CapSure) attached to a rigid support is placed in contact with the tissue section. The transfer film is activated by a near infrared laser pulse which melts the film onto the targeted cells, thus forming a strong focal bond. The desired amount of cells from the area of interest is captured onto one cap and is now ready for analysis. Conventional LCM typically uses 20-100 cells for one PCR reaction or

D

E

F

mtK14ne°

--~

IoxP neo

I

IoxP '}~~H~_~ K14.CREPR1+ RU486

IoxP FI~. 3. Induction and characterization of blisters in K14-CrePRI/mtK14 he° mice. (A) Gross phenotype of an induced blister after treatment of the right paw with inducer. No blisters developed on the untreated leg (left paw). (B) The right front paw of a K14-CrePRI/mtK14 he° pup 10 days after blister formation upon treatment. (C) The left front paw and leg of a K14-CrePR1/mtK14 he° mouse 6 months after blister formation and cessation of inducer treatment. The blistered area, including the palm and leg, healed without scarring. No additional blisters formed without further inducer treatment. (D and E) H&E staining (D) and immunofluorescence microscopy with an anti-K14 antibody (Texas Red, E) of an induced blister edge. Blistering occurred in the basal cell layer (arrowheads) of the paw. The asterisk denotes cytolysis. (F) Schematic representation of the Cre-mediated excision of the neo cassette in skin areas treated with the inducer. LCM analysis was performed on nontreated skin, on the roof of the blister in panel A, and on the blistered area after healing. [Modification of Fig. 4 in T. Cao, M. A. Longley, X. J. Wang, and D. R. Roop, J. Cell. BioL 152, 651 (2001) by copyright permission of The Rockefeller University Press.]

EBS-Model

EHK-Model

A

D

Activator

Actiyator

B

E

C

F

Phenotypically Normal Stem Cell Stem Cell with Activated K10 Mutation TA Cells and Differenting Progeny with Activated K10 Mutation FIG. 4. Schematic representation of the differences in behavior of epidermal stem ceils in our mouse models for EBS and EHK. (A, D) Stem cells in the basal cell layer express low levels of the mutant K14 allele (mtK14ne°). The mutant K10 allele (mtK10he°) is expressed at a low level in suprabasal cells. The expression of both mutant keratin alleles is insufficient to cause cell fragility, i.e., resulting in a subclinical phenotype. (B, E) On topical application of an inducer to the skin of K14-CrePR l/mtK 14he° and K14-CrePRI/mtK10 he° mice, respectively, the mutant alleles are activated by excision of the neo cassettes, thereby generating the dominant disease alleles (mtK14 l°xP, mtK101°xl'). Blisters are formed as a consequence of the activator treatment. Within a few days, lesions are reepithelializing from the surrounding epidermis (arrows). (C) Nonphenotypic stem cell (mtK14he°) colonizes the wound area in the EBS model. As a consequence, blisters heal without scarring. (F) Although the [leo cassette is excised from the mutant K10 allele, the gene is not expressed in stern cells, only in its differentiating progeny. Consequently, there is no selective pressure against stem cells containing the mtK101°~ allele. These stem cells persist and give rise to islands of mutant cells that result in persistent lesions throughout life.

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5000 cells to generate a cDNA library. 15 We routinely capture about 1000 cells per cap to perform several PCR reactions. LCM works best with stained and dehydrated sections. Any moisture, even a small amount, appears to inhibit the transfer of cells to the transfer film. In our hands, it has helped to rerinse the sections in xylene and let them air dry briefly before continuing the capturing process. 6. For DNA analysis, DNA was extracted in a buffer containing 1 mg/ml proteinase K, 0.5% Nonidet P-40, 0.25% Tween 20, 0.2 mM EDTA pH 8.0, and 10 mM Tris-HC1 pH 8.0. We usually use a volume of 50/21. The cap is placed onto a 0.5-ml tube and incubated upside down at 37 ° overnight with or without shaking. It is critical that the digestion buffer contact the tissue on the cap. 7. The next morning, the tube is centrifuged for 5 min and the cap is removed. The reaction is heated to 95 ° for 8 min to inactivate the proteinase K. An aliquot can be used directly for PCR amplification. In our experience, 40 cycles work well to amplify the sequence of interest. Conclusions Laser capture microdissection was a valuable tool in the analysis of these inducible mouse models for inherited skin blistering diseases. It allowed us to isolate contaminant-free keratinocytes from lesional and nonlesional areas of the skin and confirm at the molecular level whether Cre-mediated excision of the neo cassette had occurred. The information obtained by LCM provided new insight into the molecular and cellular basis of mosaic skin disorders, suggesting that a lack of selective pressure against stem cells containing certain mutations could explain the existence of mosaic forms of some diseases, but not others. Acknowledgments We thank C. Allred for use of the LCM equipment, E Koch and B. Eckes for help with artwork, and M. Koster for comments on the manuscript. This work was in part supported by the Deutsche Forschungsgemeinschaft (Ar 291/1-1 and Ar 291/3-1) and the Koeln Fortune Program, Faculty of Medicine, University of Cologne (M.J.A.) and NIH Grants HD25479, AR62228, and AR47898 to D.R.R.

15C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, E D. Smith, J. I. Peterson, E. Wellner, M. Ghany, and R. E Bonner, BioTechniques26, 328 (1999).

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[ 181

[181 Use of L a s e r Microdissection in Complex T i s s u e B y HOLGER S. WILLENBERG, RHODRI WALTERS,

and STEFANR. BORNSTEIN Introduction Light microscopy from a classical point of view was designed primarily to observe objects rather than to perform manipulations of cells or tissues. However, the knowledge that macroscopic phenomena originate from regulated ultrastmctural and molecular alterations led to the desire to specifically investigate single cells, or even subcellular particles and structural elements. The characteristics of light permit particles the size of micrometers to be trapped within an electromagnetic field without being mechanically or chemically altered when the light is not absorbed. On the other hand, when a parallel laser light beam is strongly focused and energy absorption at the site of interest is very high, it is possible to cut thin lesions within the tissue. At such intensities, light is, however, able to damage ultrastmctures by heat absorption causing tissue degeneration, development of plasma, or mechanical deformation. Therefore, the pulse rate and wavelength of the laser is adjustable within a certain range. Instruments designed to perform laser manipulation under visual control are now commercially available and playing an ever more prominent role in modem routine research of complex tissue.

B r i e f H i s t o r y of L a s e r M i c r o d i s s e c t i o n The development of laser microdissection shares a common history with the development of both molecular biology and microelectronics. In the late 1960s and early 1970s, the first steps were taken when chromosomal lesions were achieved using an argon laser. 1 Scientists later succeeded in dissecting tissue with precision in the range of micrometers employing a UV laser. 2,3 Unfortunately, this approach required bulky equipment and was susceptible to difficulties. Besides, it was a time-consuming procedure and, therefore, did not find its way into routine biomedical investigation. Micropipettes and needles were easier to handle and were thus used manually to scrape off tissue fragments as small as 2 square millimeters. 4,5

l M. W. Berns, R. S. Olson, and D. E. Rounds, Nature 221, 74 (1969). 2 G. Isenberg, W. Bielser, W. Meier-Ruge, and E. Remy, J. Microsc. 107, 19 (1976). 3 W. Meier-Ruge, W. Bielser, E. Remy, E Hillenkamp, R. Nitsche, and R. Unsold, Histochem. J. 8, 387 (1976). 4 M. Palkovits, Int. Rev. Cytol. 56, 315 (1979). 5 M. Palkovits, Methods Enzymol. 103, 368 (1983).


Copyright2002.ElsevierScience(USA). All fightsreserved. 0076-6879/02$35.00

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When employing a micromanipulator, however, the number of cells selected could be reduced from many hundreds to fewer than 100 cells, 6 a method which could equally well have been applied to isolate cells from pancreatic islets, acinar glands, and other structures from within paraffin-embedded tissue. However, this did not yet allow the study of single cells within complex tissues, let alone elements from single cells. In addition, this method was very limited in its reproducibility. Selective ultraviolet radiation fractionation (SURF) was described that same year. Chinese ink serves to protect DNA within the cell groups of interest from stray UV photons when irradiation of the whole tissue is performed to ablate nonprotected cells and tissue regions. 7 This technique improved reproducibility considerably. However, it did not improve biological resolution. Microdissection methods were modified and technically improved, 8-I° but remained tedious and very time-consuming. A revival of laser microdissection began with the work of Y. Kubo et al. 11 and M. Emmert-Buck et al. 12when tissue areas in the micrometer range became readily dissectable. Meanwhile laser technology became more refined and powerful, and computers became available which could run sophisticated software for controlling the laser, video processing, image acquisition, analysis, and documentation. Today some countries have funded programs for developing microdissection devices (e.g., United States, Australia), and several companies from different countries offer commercial laser microdissection systems which are user friendly. Principles UV Laser Microdissection

Laser beams with a wavelength within the ultraviolet (UV) spectrum of light are very powerful. Proteins, however (280 nm), and nucleic acids (230-260 nm) are the main energy absorbers resulting in tissue damage. Therefore this technique is used effectively as an optical scissors or knife. Laser spot widths within the range of nanometers are generated and are only used to create linear cuts. Inverted (SL Microtest, Germany; P.A.L.M. Microlaser Technologies AG, Germany; and others) and standard microscopes (Leica, Germany) are equipped with a robotized 6 L. Whetsell, G. Maw, N. Nadon, D. E Ringer, and E V. Schaefer, Oncogene 7, 2355 (1992). 7 D. Shibata, D. Hawes, Z. H. Li, A. M. Hernandez, C. H. Spruck, and P. W. Nichols, Am. J. Pathol. 141, 539 (1992). 8 R. Ktippers, M. Zhao, M. L. Hansmann, and K. Rajewsky, EMBO J. 12, 4955 (1993). 9 Z. Zhuang, E Bertheau, M. R. Emmert-Buck, L. A. Liotta, J. Gnarra, W. M. Linehan, and I. A. Lubensky, Am. J. Pathol. 146, 620 (1995). 10 j. j. Going and R. E Lamb, J. PathoL 179, 121 (1996). 11 y. Kubo, E Klimek, Y. Kikuchi, E Bannasch, and O. Hino, CancerRes. 55, 989 (1995). 12 M. R. Emmert-Buck, R. E Bonner, E D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996).

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SPECIALIZED USES

step 2

step I A

transparent cap with adhesive fil

LCM B

specimen with slide membmne +specimen

[ 18]

step 3 'aser )ean

cells of interest

slidl

s.des

CellS OTinterest

ser life"

UVpD C

visual control FIG. 1. (A) Illustrates the steps involved in laser capture microdissection (LCM). After pretreatment of the tissue on a slide, it is viewed under an inverted microscope. Step 1--A film-coated cap is placed onto the tissue. Step 2--A light spot appears which serves to aim at cells of interest. Then a laser is pulsed at the demarcated region. Step 3--This procedure is subsequently followed by the capture of the cells which stick to the cap, which is then removed and brought in contact with prepared solutions. (B) Illustrates the principle of UV laser microdissection (UV #D). Specimens are prepared on a membrane holder which is supported by a slide. On the video screen, one can draw projected laser lines with a computer to program the laser and the robotized microscope stage. Subsequently, the dissected structures can be removed mechanically with a sticky cap or they fall into microcentrifuge tubes. (C) Gives an example of tissue (adrenal) before and after laser microdissection.

stage and controlled by a computer. A n i m a g e is displayed on a v i d e o screen. S p e c i m e n s are placed on holders or slides w h i c h are c o v e r e d with a specialized m e m b r a n e and can be subjected to pretreatment in the same m a n n e r as glass slides, with applications such as histochemistry, paraffin removal, or i m m u n h i s t o l o g i c a l procedures. T h e cells or areas to be excised are m a r k e d on the v i d e o screen using a m o u s e in the same w a y as m o d e m graphic p r o g r a m s are used to draw lines, circles, etc. T h e n the robotized m i c r o s c o p e - o p e r a t e d laser and stage are used to cut out the m a r k e d objects as per prior c o m p u t e r generated circumscription (Fig. 1). The dissected tissue fragments are collected automatically in m i c r o c e n t r i f u g e vials, or else m e c h a n i c a l l y through m i c r o c e n t r i f u g e lids coated with an adhesive film that b e c o m e s activated by the laser energy. Since the samples are protected by a m e m b r a n e , they can be transferred without being touched.

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UV lasers are also employed as "zona knives," for example, to dissect the zona pellucida of mammalian oocytes, or as "nano knives," for instance, to cut the tails of sperm cells. UV laser microdissection (UV #D) can even be used to perform incisions into subcellular structures such as membranes. Thus, for example, in vitro fertilization is facilitated using UV laser microdissection techniques. Laser Capture Microdissection This method, developed at the National Cancer Institute (NCI), NIH, Bethesda, MD, with the company Arcturus, Mountain View, CA, makes use of a low energy infrared (IR) laser whose energy is not absorbed by proteins and is too low to heat water over a point at which tissue destruction could result. 12 A high precision inverted microscope provides visual control over the tissue to be dissected. A sterile film-coated cap is applied to the top of the tissue, and an area of interest is chosen. A laser penetrating the cap is triggered under optical control. The film is an inert membrane and melts under heat, becoming glue-like and sticking to the tissue area on which the laser impinges (Fig. 1). Because of the low energy and the short pulse duration of the laser beam, almost no damage is done to the structures to be investigated. Nevertheless, the ceils are subjected to heat as well as photons from the laser itself which may result in partial destruction of nucleic acids. After successful dissection, the cap can be removed and transferred to a 500-/zl microcentrifuge tube or a special container which allows a minimal volume of appropriate lytic solution to be added to extract the material from the microdissected cells. The laser spots are so small that even single cells can be isolated from slides carrying quite complex tissue structures. This procedure is very fast and has therefore been integrated into the Cancer Genome Anatomy Project (CGAP) of the National Cancer Institute at NIH.

Applications Visually controlled microdissection techniques combine the advances of anatomical and physiological approaches. The optical contrast achieved by histologic pretreatment of the specimen and dissection of the tissue under fine visual control, in conjunction with the added resolution of molecular studies with emphasis on genetics and proteomics, result in a deeply impactive augmentation of biofunctional resolution. Thus, laser microdissection affords a wide range of applications. Paraffin-embedded samples can be processed as well as frozen tissue, cell smears, and even cell culture samples. New histological procedures improve optical resolution, particularly when special membranes are employed to select and collect samples directly from cryo-microtomes. Histochemistry and immunohistochemistry can be performed prior to microdissection to mark or highlight objects of interest. Thus, the contrast between benign and malignant cells, or the

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contrast between specialized organ-specific cells and cells belonging to other tissues, e.g., nerve cells, endothelial cells, infiltrating immune cells, or cells from within complex tissues, can be enhanced. The clinical function of a whole organ results from a complex interaction between various cell types and the development of the ultrastructure within which these cells are embedded. Dependent on their age, their exposure to changes within the micromilieu, or their localization within the tissue, cells of the same type and origin may fulfill differing functions or undergo further differentiation. Adrenal chromaffin cells, for instance, may develop into a sympathetic neuron-like cell type or else into a neuroendocrine type which produces mainly catecholamines 13 under the influence of adrenocortical glucocorticoids. 14,15 Chromaffin cells also regulate the function of adrenocortical cells. 16,17 W h e n grown in coculture with medullary cells, adrenocortical cells increase their cortisol production as much as tenfold as compared to adrenocortical cells maintained in monoculture. TM Using microdissection systems it is now much easier to achieve molecular a c c e s s to understanding these paracrine interactions. Structures can now be separated via laser knives and be subjected to downstream analytical methods without fear of contamination by surrounding tissue. This is even more important in organs built up by various types of cells which intermingle to such a great degree as in the adrenal gland. Thus, for instance, it is possible to examine the differential expression of the receptor for prolactin and the receptor for leptin within the three zones of the adrenal cortex, in the adrenal medulla, and also in tumorous adrenal tissue. 19,2° In a clinical case of Cushing's syndrome, which arose due to ectopic adrenal ACTH secretion, an adrenocortical-pituitary hybrid tumor was identified using L C M followed by combined molecular and ultrastructural analysis. 21 Studies concentrating on spatial resolution can be performed as well as studies which aim at improving temporal resolution, focusing on development, regeneration, and remodeling of tissues, on cell proliferation or necrosis and apoptosis. 13D. J. Anderson and R. Axel, Cell 47, 1079 (1986). 14R. J. Wurtman and J. Axelrod, Science 150, 1464 (1965). 15D. P. Merke, G. P. Chrousos, G. Eisenhofer, M. Weise, M. E Keil, A. D. Rogol, J. J. Van Wyk, and S. R. Bornstein, N. Engl. J. Med. 343, 1362 (2000). 16M. Ehrhart-Bornstein,J. P. Hinson, S. R. Bornstein, W. A. Scherbaum, and G. P. Vinson, Endocr. Rev. 19, 101 (1998). 17S. R. Bornstein, H. Tian, A. Haidan, A. Bottner, N. Hiroi, G. Eisenhofer, S. M. McCann, G. P. Chrousos, and S. Roffler-Tarlov,Proc. Natl. Acad. Sci. U.S.A. 97, 14742 (2000). 18A. Haidan, S. R. Bornstein, A. Glasow, K. Uhlmann, C. Lubke, and M. Ehrhart-Bomstein, Endocrinology 139, 772 (1998). 19A. Glasow,S. R. Bornstein, G. P. Chrousos, J. W. Brown, and W. A. Scherbanm,Horm. Metab. Res. 31, 247 (1999). 20A. Glasow, A. Haidan, U. Hilbers, M. Breidert, J. Gillespie, W. A. Scherbanm, G. P. Chrousos, and S. R. Bornstein, J. Clin. Endocrinol. Metab. 83, 4459 (1998). 21N. Hiroi, G. P. Chrousos,B. Kob_n,A. Lafferty,M. Abu-Asab,S. Bonat, A. White, and S. R. Bornstein, J. Clin. Endocrinol. Metab. 869 2631 (2001).

[1 8 ]


22 1

This remains one of the key foci of the CGAP project at NIH. Genetic fingerprints of various tumors at different stages of progression and with different malignant characteristics are compiled within a huge gene library and then catalogued (cgap.nci.nih.gov/). 22,23 Thus, it was possible to identify the mutations and the gene responsible for the development of multiple endocrine neoplasia (MEN) type 1.24 To examine somatic mutations there is a special need to separate with high precision stromal cells from neighboring cells which derive from the endothelium, from nervous tissue, or from fibroblasts. In using preparations derived from single cells, it is possible to detect a single point mutation in the codon 12 of the c-Ki ras 2 mRNA. 25 The study of identified proteins or mRNAs expressed within malignant tissue and a direct comparison of the tissue structure to the entries within the gene library resulted in the discovery of genes which are excusively expressed in prostate cancer. 26 New concepts which study the aberrant expression or overexpression of proteins in context of tumor developmentz7-29 led to the identification of ectopic receptors for cytokines3° or other trophic factors such as hormones and neuropeptides. 31 Tissue infiltration by immune or metastasized cells may lead to other metabolic alterations or consequences for neighboring cells and the establishment of alternative feedback systems. It was in 1889 when Stephan Paget created the hypothesis of "seed and soil" and recognized the role of tissue that embedded the tumor in oncogenesis. 32'33 A hundred years later it is now possible to have a closer look at early observed phenomena by piercing into the underlying molecular mechanisms with laser microdissection technology.

22 R. L. Strausberg, J. Pathol. 195, 31 (2001). 23 j. W. Gillespie, M. Ahram, C. J. Best, J. I. Swalwell, D. B. Krizman, E. E Petricoin, L. A. Liotta, and M. R. Emmert-Buck, CancerJ. 7, 32 (2001). 24 S. C. Chandrasekharappa, S. C. Guru, P. Manickam, S. E. Olufemi, E S. Collins, M. R. EmmertBuck, L. V. Debelenko, Z. Zhuang, I. A. Lubensky, L. A. Liotta, J. S. Crabtree, Y. Wang, B. A. Roe, J. Weisemann, M. S. Boguski, S. K. Agarwal, M. B. Kester, Y. S. Kim, C. Heppner, Q. Dong, A. M. Spiegel, A. L. Burns, and S. J. Marx, Science 276, 404 (1997). 25 K. Schiitze and G. Lahr, Nat. Biotechnol. 16, 737 (1998). 26 G. Vasmatzis, M. Essand, U. Brinkmann, B. Lee, and I. Pastan, Proc, Natl. Acad. Sci. U.S.A. 95, 300 (1998). 27 M. S. Katz, T. M. Kelly, E. M. Dax, M. A. Pineyro, J. S. Partilla, and R. I. Gregerman, J. Clin. Endocrinol. Metab. 60, 900 (1985). 18 A. Lacroix, E. Bolte, J. Tremblay, J° Dupre, P. Poitras, H. Fouruier, J. Garon, D. Garrel, E Bayard, R. Taillefer, R. J. Flanagan, and P. Hamet, N. Engl. J. Med. 327, 974 (1992). 29 y. Reznik, V. Allali-Zerah, J. A. Chayvialle, R. Leroyer, P. Leymarie, G. Travert, M. C. Lebrethon, I. Budi, A. M. Balliere, and J. Mahoudeau, N. Engl. J. Med. 327, 981 (1992). 30 H. S. Willenberg, C. A. Stratakis, C. Marx, M. Ehrhart-Bornstein, G. P. Chrousos, and S. R. Bornstein, N. Engl. J. Med. 339, 27 (1998). 31 N. N'Diaye, J. Tremblay, P. Hamet, and A. Lacroix, Horm. Metab. Res. 30, 440 (1998). 32 S. Paget, Lancet 1, 571 (1889). 33 G. Poste and L. Paruch, CancerMetast. Rev. 8, 93 (1989),

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pretreatment (immuno-)histology • in v i v o tracing • in v i t r o labelling

laser microdissection

•

mutational analysis I

[ 18]

quantitative PCR

cDNAlibraries

nucleic acids, proteins, particles

microarrays proteomics, MALDI, etc.

II

III

FIG. 2. Schematic flow of the combination of methods possible employing laser (capture) microdissection systems.

Laser microdissection is thus not only a tool for "digging into unknown ground" and identifying factors or receptors implicated in tumorigenesis, but also capable of excluding peptides which were believed to play a major role in these processes. For example, authors were able to discount the hypothesis that mutations in the gene coding for the prolactin receptor were a pathophysiologicaUy relevant step in the development of human breast carcinomas, an Perspectives The classical experimental sequence--isolation of a "specific" cell, amplification of its genome, characterization of its mRNA expression profile, identification of characteristics which lead to its specialized role is no longer a scientist's dream today (Fig. 2). Purified preparations of nucleic acids can now be hybridized to one of thousands of gene probes on a microchip array. 35 Proteins can be denatured or maintained on a slide and analyzed by applying the protein on antibody arrays that recognize selective epitopes, or else be analyzed within a matrix-assisted laser desorption device or by ionization-time-of-flight mass spectrometry devices, with many more technologies in development within the new wave of proteomics. 34 A. Glasow, L. C. Horn, S. E. Taymans, C. A. Stratakis, E A. Kelly, U. Kohler, J. Gillespie, B. K. Vonderhaar, and S. R. Bornstein, Z Clin. Endocrinol. Metab. 86, 3826 (2001). 35 M. A. Rubin, J. Pathol. 195, 80 (2001).

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Aspiration of biological material, laser microdissection of diseased structures, and subsequent analysis using modern techniques of molecular medicine represent a combination of methods which will be in routine use in the years ahead in diagnostics, development of therapeutical agents, and monitoring treatment. Summary Concomitant with the rapid development in biomedical knowledge, including the methods of molecular biology and proteomics, and the manufacture of ever more precise optical instruments, powerful lasers, and sophisticated microcomputing hardware and software, laser microdissection systems have emerged which are now entering the field of routine research. Today, several devices are commercially available, congresses devoted to the latest advances in laser microdissection are now held on regular occasions, and the number of publications based on the use of these techniques has risen to over 250. With laser microdissection, histological treatment, such as chemical or immunological fixation and staining, can readily be combined with methods suitable for molecular biology or proteomics. As the optical, technical, and methodological resolution of polymerase chain reaction (PCR) and microdissection increases, genetic and phenotypic studies of biological material are possible even at the level of single cells and subcellular elements. Moreover, questions such as the paracrine interaction of cells within complex tissues, the development of cancer, and the role of single cells in tissue remodeling or development on the microscopic and molecular level can now be addressed precisely at the molecular level. This chapter reviewed the development of laser microdissection platforms, its potential impact on the future of research, and how, in particular, these technologies can be successfully integrated into modem research and routine histopathological studies of complex tissue. Acknowledgment This work was supported by a DFG grant (441/i-1 to S.R.B). We would like to express our thanks to Dr. J. Gillespie and the NIH LCM Lab for technical support and advice.

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[191 Assessment of Clonal Relationships in Malignant Lymphomas By KOJO S.

J. ELENITOBA-JOHNSON

Introduction The recent development of new technologies that provide the opportunity for direct molecular examination of pathologic cells and tissues has dramatically transformed investigative studies in research and diagnostic medicine. These techniques permit exploration and analysis of molecular and cellular aberrations as well as of mechanisms of development of disease processes. One technology that has found widespread utilization for the isolation of specific cell populations is laser capture microdissection (LCM)J LCM is a recently developed technology that involves microscopic visualization and isolation of defined cell populations from tissue sections. The histologic area of interest on the tissue section is visualized and directly overlaid by a specialized cap on which a transparent ethylene vinyl acetate thermoplastic film is coated. The thermoplastic polymer film contains infrared (IR) absorbing dyes that permit utilization with near-IR gallium arsenide laser diodes, which have been incorporated into routine microscopes. Operatordependent pulsed laser activation effects transient melting of the thermoplastic polymer in the vicinity of the targeted cells. The cells are captured within the film and can be retrieved in aqueous solutions for subsequent experimental manipulation. The commercially available versions of the LCM instruments permit modulation of the spot size and laser beam intensity. The transient temperature transitions encountered in the process are innocuous to nucleic acids in the tissues, and DNA and RNA can be easily recovered for utilization in experiments. The unique property of lymphoid cells to undergo specific rearrangements of their antigen receptor genes (ARG) within a virtually infinite repertoire of combinatorial diversity provides a convenient modaiity of assessment of clonality. The assessment of clonal relationships between two or more putative neoplastic populations is a useful strategy in the determination of the origin or the relationships between anatomically or temporally separate populations. The advent of LCM has dramatically improved the ability to assess clonal relationships, by permitting tumor cell purification or enrichment, and in some cases, isolation of single cells at the histological level. Once tumor cell enrichment or isolation of pure tumor populations is accomplished, it is feasible to evaluate clonal relationships by examining for genetic markers characteristic of the particular neoplasm of interest. 1 M. R. Emmert-Buck, R. E Bonner, P. D. Smith, R. E Chuaqui, Z. Zhuang, S.R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996).


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The ability to directly assess lymphoid populations in this fashion has had important implications for the primary distinction of malignant from benign processes, molecular staging, and monitoring of minimal residual disease following therapy. In this chapter, a number of strategies and protocols that have been successfully implemented in our laboratory for the establishment of clonal relationships between two microdissected lymphoid populations will be described. Amplification of the ARG in benign lymphoid populations yields a polyclonal smear or ladder pattern on gel electrophoresis indicating the presence of recombined antigen receptor genes of different lengths. On the other hand, a malignant population arising by clonal expansion of a neoplastically transformed lymphoid cell exhibits only one or two specific gene rearrangements. This antigen receptor rearrangement is unique to the founder neoplastic cell and its progeny and thus polymerase chain reaction (PCR) analysis of ARG in a monoclonal population yields either a single or two prominent bands. This unique rearrangement serves as a marker for this particular neoplasm and can be used as a parameter for the establishment of a clonal relationship between this and another lymphoid population. In this regard, the reader will note that all of the examples illustrated in this section relate to B-lymphoid neoplasms. Similar principles apply to T-cell processes in which the T-cell receptor F or fl genes may be amplified for the establishment of clonal relationships between two or more microdissected T-cell populations. While the strategies and protocols discussed here are by no means exhaustive, they provide a framework for the design of similar assays based on parallel fundamental concepts. Protocols

Preparation of Fixed Paraffin-Embedded Tissue Sections for Laser Capture Microdissection Unstained 5/~m thick sections are deparaffinized by dipping into xylene, rinsed twice with 95% ethanol, briefly stained with eosin, and air-dried.

Preparation of OCT-Embedded Frozen Tissue Sections for Laser Capture Microdissection Five-micron sections are cut from OCT embedded snap-frozen tissue blocks using a cryostat. The sections are mounted on poly-L-lysine precoated glass slides (C to C Laboratory Supplies, Chicago, IL). The frozen sections are thawed at room temperature for 60 sec and submerged in either cold acetone (5 rain), methanol (5 min), 4% paraformaldehyde (5 min), 70% ethanol (30 sec), ethanol/formalin (3 : 1, 1 min), and ethanol 70% (15 sec) followed by acetone (5 min). The slides are then briefly rinsed in phosphate buffered saline (PBS), pH 7.4, and allowed to

dry.

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Imrnunohistochemical Staining for Laser Capture Microdissection Immunohistochemical staining is performed using a three-step streptavidinbiotin technique with monoclonal or polyclonal (rabbit anti-human) antibodies against the appropriate antigen that is specific for the intended cell population, and the DAKO Quick Staining kit (DAKO Corp., Carpinteria, CA). The slides are incubated at room temperature with the primary and secondary antibodies, and the tertiary reagent for 90 sec each, and rinsed with 1 × PBS between each step. Color development is performed using diaminobenzidine (DAB) (3 min) as the chromogen. The sections are counterstained with hematoxylin for 15 sec (optional), serially dehydrated through graded alcohols (3 dips in each alcohol solution), and finally immersed in xylene before air-drying at room temperature. After drying, the immunohistochemically stained section is ready to be utilized for LCM.

Laser Capture Microdissection Laser capture microdissection is performed using a PixCell laser capture microscope (Arcturus Engineering, Santa Clara, CA). Briefly, 5 /zm hematoxylin and eosin stained tissue sections obtained from fixed paraffin-embedded or frozen OCT-embedded tissues are placed under the microscope and the area of interest is focused between the optical cross hairs visible in an eyepiece lens. The optically clear plastic caps bearing the thermoplastic membranes (which effect the capture of cells from the glass slides) are loaded onto the transporting arm attached to the microscope. The transporting arm with the loaded plastic cap is swiveled across to overlay the plastic cap on the area of interest on the glass slide (Arcturus Engineering, Santa Clara, CA). Laser activation of the film leads to focal melting at the precise spot of activation with attendant capture of the underlying visually selected cells (Fig. 1). The cell-laden cap may now be utilized as the cover for the Eppendorf tube that contains a previously aliquoted volume (~20/zl) of the digestion buffer. The tube is inverted gently and repeatedly to immerse the microdissected cells within the digestion buffer. Brief pulses of vortexing may be performed to facilitate dissolution of the cells into the digestion buffer.

DNA Extraction Approximately 500 cells from each biopsy specimen are procured by microdissection and immediately transferred into a 20/zl digestion buffer solution containing 0.05 mol/liter Tris-HCL, 0.001 mol/liter EDTA, 1% Tween 20, and 0.1 mg/ml proteinase K (pH 8.0) and incubated overnight at 37 °. Alternatively, microdissectates may be incubated at 55 ° for 3-5 hr. In either case, the mixture is heated to 95 ° for 10 min to inactivate proteinase K, and 1.0/zl of this solution is used as the template for PCR. Further purification of the DNA can be achieved

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FIG. 1. Illustration of microdissection of two histologically distinct lymphoid populations. (A) I00 × (original) magnification of a hematoxylin and eosin stained section showing follicular lymphoma with attendant monocytoid B-cell proliferation before microdissection. (B) After microdissection of both follicular and monocytoid B-cell components.

in some cases by pursuing the additional steps of phenol-chloroform extraction and ethanol precipitation. In our hands, we have found that the crude proteinase K digests yield adequate DNA material for PCR. The integrity of the DNA from the microdissected sample may be established by successful PCR of a segment of a housekeeping gene (e.g., human/~-globin). During the configuration of the housekeeping gene PCR, the product size should be comparable to or greater than that of the intended gene of interest. We have successfully verified the integrity of isolated DNA from frozen and paraffin-embedded tissue material using primers from the human hemoglobin beta chain gene. 2 These primers yield 123 bp and 268 bp PCR products and can be readily visualized by ultraviolet transillumination of ethidium bromide-stained (0.2/zg/ml) 1.5% NuSieve agarose gels (Nusieve 3 : 1 Agarose blend, FMC BioProducts, Rockland, ME). On occasion, the 123 bp may be present, but the 268 bp fragment may absent. This is interpreted as evidence that the extracted DNA is adequate for amplifying only smaller DNA fragments. For instance, such a sample would be considered inadequate for immunoglobulin

2 G. Wu, T. C. Greiner, and W. C. Chang, Diagn. MoL Pathol. 6, 147 (1997).

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[ 191

heavy chain (IgH) PCR using framework II primers which typically yield products of approximately 250 base pairs.

Assessment of Clonal Relationships by Polymerase Chain Reaction Analysis of lmmunoglobulin Heavy Chain Gene Polymerase chain reaction assays to detect IgH gene rearrangements exploit conserved sequences with!n the variable and joining regions of the IgH gene that are located several kilobases apart in the germ-line configuration. In B cells that have undergone IgH gene rearrangement, these variable and joining regions are brought into close proximity. No product is amplified in nonlymphoid cells, whereas B cells yield short PCR products whose length and sequence composition is determined by the nature of their recombined variable-diversity-joining (V-D-J) regions and by their unique N (nontemplated nucleotide) region sequences. Importantly, because of the tremendous repertoire for diversity present at the antigen receptor loci, simultaneously tested lymphoid samples with IgH PCR products of identical sequence may be presumed to be clonally identical. Although the unique IgH complementarity determining region (CDR) sequence of a monoclonal lymphoid population is the gold standard for assessing clonal relatedness, it is also reasonable to surmise that samples demonstrating IgH PCR products of identical size are clonally related (Fig. 2). A similar interpretation holds for T-cell receptor y chain gene PCR assays for the establishment clonal relationships in T-cell populations.

IgH PCR For IgH heavy chain PCR, crude extract (1 or 2/xl) from microdissected tissue and 1 /zl each of 50 ng/#l positive and negative controls are used as the template for amplification. PCR is performed in a PerkinElmer 2400 thermal cycler (PerkinElmer, Norwalk, CT). Target DNA is amplified in a 20/zl reaction containing GenAmp PCR buffer, 1.5 mM MgC12, four deoxynucleotide triphosphates at 200/xM, primers at 0.5/zM, and 1 unit of AmpliTaq DNA Polymerase (PerkinElmer). Labeled reactions include a 32p dCTP, and the concentration of cold nucleotides is reduced (50 IzM dATP, dGTP, and dTTP; 10/zM dCTP). A hot start technique is employed using TaqStart Antibody (Clontech, Palo Alto, CA), and immunoglobulin heavy chain variable region (VH-FRIII); 5'-ACA CGG C[C/T] T[G/A]T ATT ACT GT-3') and joining region (JH; 5'-ACC TGA GGA GAC GGT GAC C-3') consensus primers. The amplification protocol consists of 35 cycles of denaturation at 96 ° for 1 min, annealing at 56 ° for 1 min, and extension at 74 ° for 1 min. Amplification products may be analyzed on ethidium bromide stained standard polyacrylamide or sequencing gels, with molecular weight standards. Radiolabeled products are best analyzed on 8% sequencing gels, dried and autoradiographed. One or two dominant bands indicate the presence of a monoclonal

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120 bp

FIG. 2. Immunoglobulin heavy chain PCR from two microdissected lymphoid populations analyzed on a 10% polyacrylamide gel. Lane 1 corresponds to the H20 control. Lane 2 corresponds to the positive control (Raji cell line). Lanes 3--4 represent duplicate IgH PCR amplifications from one microdissected population, and lanes 5-6 contain products from the other component. There is comigration of monoclonal bands indicating that the two different lymphoid populations are clonaUy identical.

population, whereas a polyclonal population results in either a smear (standard acrylamide gel) or a ladder of bands of nearly equal intensity (sequencing gel). Replicates of all PCR-based gene rearrangement analyses of microdissected samples should be run parallel in order ensure reproducibility and to avoid erroneous scoring of monoclonality as a result of low lymphocyte numbers. 3 Isolation of Monoclonal Bands for Direct DNA Sequencing

Amplicons resulting from IgH PCR can be subjected to electrophoresis using ethidium bromide-stained 1.5% agarose gels, and the band of expected size excised, dissolved in distilled H20, purified, and concentrated using Amicon DNA Extraction columns (Millipore, Bedford, MA), according to manufacturer's instructions. A denaturation gel-based approach has been suggested by Wu et al., 2 which facilitates ready distinction of monoclonal bands. This method requires the utilization of GC-clamped IgH V region primers. The GC clamp portions of the GC-clamped amplicons derived from the IgH PCR may be used as priming sites for direct DNA sequencing.

3 K. S. Elenitoba-Johnson, S. D. Bohling, R. S. Mitchell, M. S. Brown, and R. S. Robetorye, J. Mol. Diagn. 2, 92 (2000).

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Direct Purification of PCR Amplicons for Cycle Sequencing The generation of optimal DNA sequence requires elimination of excess primers and dNTPs that remain following PCR amplification. We have utilized the QIAquick PCR purification kit (Qiagen, Valencia, CA) with good success. The purification is based on column technology that utilizes the selective binding properties of silica-gel membranes. The silica membrane adsorbs DNA in the presence of high concentrations of chaotropic salts and optimal pH. Adsorption is typically 95% at pH < 7.5. The purified DNA is eluted with distilled water or Tris buffer. We have observed that lower salt concentrations and basic conditions (pH 7.5-8.5) generally enhance elution efficiency. Ten replicates of the sample of interest are amplified and pooled. Five volumes of Buffer PB are added to 1 volume of the pooled PCR product. The samples are the applied to the wells of the QIAquick strips and the vacuum source is switched on. After all the liquid has been suctioned through the column, the vacuum is switched off and the QIAquick 6S is vented by releasing the tubing connecting the unit to the vacuum. The wells of the QIAquick strips are washed by adding 1 ml of the Buffer PE to each well and then the vacuum switched on. For optimum washing, the last two steps are repeated with 2 washes of Buffer PE. After Buffer PE has been drawn through the columns, the membrane is dried by applying the maximal vacuum for an additional 5 min. After this step, the vacuum is switched off and the QIAquick 6S is ventilated. The top plate is disengaged from the base and the nozzles of the QIAquick strips are blotted with clean Kimwipes. The DNA is eluted by adding 75-100 #1 of Buffer EB (10 mM Tris-HC1, pH 8.5) directly to the center of each membrane in the QIAquick strips and allowed to stand for 1 min. The vacuum is switched on for 2 min to effect suction through the QIAvac 6S. Samples in which sequencing is to be performed in the short term (

5O"

Leptin

1/i IL-6

FIG. 3. (A) RT-PCR analysis of the effects of TFGfll and PACAP-38 on leptin and IL-6 mRNA expression in pituitary FS cells. Cultured cells are collected by immuno-LCM, analyzed by RT-PCR, and normalized with HPRT. Lane 1, Control pituitary FS cells; lane 2, TGF/~ 1-treated FS cells; lane 3, PACAP-38-treated FS cells; lane 4, normal rat pituitary cells without LCM, used as a positive control; lane 5, normal rat pituitary cells without RT, used as a negative control. The toppanel in A shows the ethidinm bromide-stained gel; the bottom panel of A shows Southern hybridization with the internal probe described in Materials and Methods. (B) Densitometric analysis show a 1.8-fold increase in leptin mRNA by TGFfll treatment. Data are the mean q- SEM from three experiments. Reproduced from L. Jin, I. Tsumanuma, K. H. Ruebel, J. M. Bayliss, and R. V. Lloyd, Endocrinology 142, 1703 (2001) with permission from the Endocrine Society.

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the TtT/GF cell line, which may be related to the rat primers used. PRL, GH, and POMC mRNA were detected in the normal pituitary, but not in the TtT/GF cells.

TGF~I Regulation of Leptin mRNA Expression After treatment of dissociated pituitary cells with TGF/~ 1 or PACAP-38 followed by immuno-LCM, RT-PCR, and Southern hybridization, there is increase in leptin, but not IL-6, mRNA expression (Figs. 3A and 3B). 26 This effect is specific for TGF~ 1, as PACAP-38 did not influence leptin or IL-6 mRNA expression.

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ANALYSIS OF GENEEXPRESSION

259

[22] Analysis of Gene Expression By JANETTEK. BURGESS and BRENT E. MCPARLAND

Introduction The recent advances of the collaborative sequencing programs that are decoding the complete genomes of many organisms are providing opportunities for new approaches to gene analysis. With these developments have come greater needs for attention to the source material to ensure that the gene expression profiles reflect the gene activity in the cells of interest rather than an experimental artifact. The advent of techniques that allow the manipulation of genetic material has significantly improved the understanding of cellular events, but the study of crude tissue extracts is complicated by the heterogeneous nature of their cellular components. The development of laser capture microdissection (LCM) has enabled the isolation of populations of defined single cell types which can then be analyzed for their DNA, RNA, or protein content (discussed in [14], this volume). 1 The ability to isolate and analyze specific individual cells from a complex multicellular sample reduces the opportunity for nonspecific contribution from colocalized cell types and will enhance our understanding of the role played by each cell type. LCM-based analysis is applicable to any disease process for which histopathological lesions are accessible through tissue sampling or other sources, e.g., cytospins of blood. Examples include mapping the field of genetic changes associated with the progression of premalignant and malignant cancer lesions; analysis of gene expression patterns in atherosclerosis, inflammation, Alzheimer's disease plaques, multiple sclerosis, and infectious microorganism diagnosis; and analysis of genetic abnormalities in utero from selected rare fetal cells in maternal fluids. 2 The ability to isolate defined cell populations from small tissue sections enables the utilization of biopsy samples for analysis of gene expression. The identification of gene expression patterns may provide vital information for the understanding of the disease process and may contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with defined pathological lesions may provide opportunities for new therapeutic targets in the future. Sample Preparation Particular attention must be paid to the pretreatment of samples from which RNA, and to a lesser degree DNA, will be isolated downstream. RNA of reasonable I K. K. Jain, Methods Enzymol. 356, [14], 2 0 0 2 (this volume).

t R. F. Bonnet,M. Emmert-Buck,K. Cole, T. Pohida,R. Chuaqui, S. Goldstein,and L. A. Liotta, Science 278, 1481 0 9 9 7 ) .

METHODSIN ENZYMOLOGY,VOL. 356

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integrity has not been successfully isolated from paraffin embedded tissues. The treatment of the tissue between tissue resection and the freezing of fresh frozen specimens is important. Tissue pH can play a critical role in the integrity of the RNA. 3,4 Successful isolation of intact mRNA in the brain is associated with a tissue pH between 6.1 and 7.0. Tissues with a pH below 6.0 result in fragmented or absent RNA. After mounting in a protective medium such as OCT (Miles, Elkhart, IN) and snap freezing in liquid nitrogen cooled isopentane or hexane, tissues should be stored at - 8 0 ° . The freshly frozen tissues should be brought to - 2 0 ° and immediately cut on the cryostat. The optimal thickness of the sections varies for different tissue types but is generally between 4 and 10/~m. The sections are placed on a plain glass slide at room temperature and immediately fixed. Pretreatment of the glass slides to remove any RNases present either by prebaking at 230 ° for 4 hr5 or by treating with RNase Away (Molecular BioProducts, San Diego, CA) helps to reduce further RNA degradation. The blade of the cryostat should be changed and the blade holder cleaned with acetone between each different tissue sample. Once sectioning of the tissue commences, it is important to work rapidly through to completion of the staining protocol to avoid RNA degradation. The integrity of the RNA within a tissue section can be checked by staining one section with 10 lzg/ml acridine orange in 0.2 M dibasic sodium phosphate/0.1 M citric acid (pH 4.0) using the method described by Ginsberg and colleagues. 3'6

Fixation and Staining Protocol All solutions used throughout the fixation and staining procedures should be prepared in diethylpyrocarbonate (DEPC)-treated water and refreshed between staining sections from a different tissue sample. Sections are fixed in 70% ethanol at room temperature for 2 to 4 min and rinsed rapidly in DEPC-water. Mayer's hematoxylin is applied to the slide surface (200 #1 per section) for 1 to 2 min followed by a rapid rinse in DEPC-water. Thirty seconds in Scott's blueing solution enhances the staining of the nuclei followed by 30 sec in DEPC-water and then 30 see in 70% ethanol. Eosin stain (500 mg/dl, alcohol soluble) is then applied to the slide surface (200 ~1 per section) for 1 min. The sections are then dehydrated through 95% ethanol for 30 see, followed by two changes of molecular sieve dried 100% ethanol for 1 min each before a final dehydration step in two changes of 3 S. Balm, S. J. Augood, M. Ryan, D. G. Standaert, M. Starkey, and P. C. Emson, J. Chem. Neuroanat. 22, 79 (2001). 4 A. E. Kingsbury, O. J. Foster, A. E Nisbet, N. Cairns, L. Bray, D. J. Eve, A. J. Lees, and C. D. Marsden, Brain Res. Mol. Brain Res. 28, 311 (1995). 5 y. Kohda, H. Murakami, O. W. Moe, and R. A. Star, Kidney Int. 57, 321 (2000). 6 S. D. Ginsberg, E B. Crino, V. M. Lee, J. H. Eberwine, and J. Q. Trojanowski, Ann. Neurol. 4L 200 (1997).

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xylene for 2 min each. Care should be taken to ensure that the tissue is not left in the xylene for too long as the tissue becomes so dry that nonspecific cell adhesion to the caps occurs. The molecular sieves should be pretreated by washing in DEPCwater for three changes of 30 min each followed by baking at 160 ° for 5 days to remove RNases. Once stained, the sections can be wrapped in aluminum foil and placed in an airtight container in the presence of silica gel. Storage time for stained sections prior to cell capture varies for different tissue types as prolonged storage can lead to irreversible adherence to the slide surface. In our experience airway smooth muscle cells from human lung sections bind to the slide surface in an irreversible manner after 72 hr, whereas the surrounding cell types are easily captured after weeks in storage. In contrast, mouse brain sections can be stored for prolonged periods and all cell types can still be captured. Alternative Fixatives

Fixation methods that alter the three-dimensional structure of the proteins within the tissues (such as ethanol, methanol, or acetone) allow the isolation of RNA from captured cells. The stronger fixation agents (such as formalin and paraformaldehyde) that cross-link the proteins within the tissues inhibit the dissociation of the tissues in the RNA lysis buffer and degrade the RNA. 7 A method of fixing tissues using methacarn prior to paraffin embedding has been described that yielded half the quantity of RNA that could be isolated from fresh frozen tissue but the total RNA integrity was well preserved. 8 The DNA contamination in this preparation was also reduced compared to fresh frozen tissue. This method of fixation may allow for subsequent paraffin embedding of tissues while still providing the option of RNA retrieval at a later stage. One other potential problem with RNA retrieval from paraffin embedded tissue is that large RNA molecules may fail to be retrieved, but further studies are needed to confirm this effect. 7'8 Alternative Stains

Many stains work only on tissues that have been prepared in a specific manner. One step often used is mordanting of tissue prior to staining. A commonly used fixative with mordanting properties is Bouin's solution which requires treatment for 1 hr at 60 °. This step is crucial for trichrome stains; therefore they cannot be used on fresh frozen tissue that has been fixed in 70% ethanol as required for the isolation of RNA. Periodic acid Schiff (PAS) staining has been observed to lead to significant RNA degradation. 5

7 S. M. Goldsworthy,E S. Stockton, C. S. Trempus,J. E Foley,and R. R. Maronpot,Mol. Carcinog. 25, 86 (1999). 8M. Shibutani, C. Uneyama,K. Miyazaki,K. Toyoda,and M. Hirose,Lab. Invest. 80, 199 (2000).

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Stains that have been reported to differentiate cell types that facilitated the isolation of quality RNA include toluidine blue, 9 cresyl violet, 8 and methyl green. 3

Identification of Cells of Interest Familiarity with the morphology of the tissue being examined is essential, as LCM requires identification of the cell types of interest in the tissue sections without the aid of a coverslip. In some cases it is not possible to distinguish between cell types without the use of cell surface markers, so rapid immunostaining techniques have been developed which enable the identification of cells while preserving the integrity of the RNA in the cells of interest. Fend and colleagues 1° have described a method of immunostaining that requires between 12 and 25 min for the complete staining protocol, depending on the nature of the primary antibody. The slides are incubated with the primary, secondary, and tertiary antibodies for 90 to 120 sec each at room temperature with 1x PBS wash between each step. Diaminobenzidine is used for 3-5 min for color development before counterstaining with hematoxylin for 15 to 30 sec and dehydration in graded alcohols (15 sec each) and xylene (twice for 2 min). The final step is to air-dry the slides before LCM is commenced. The antibodies in the Dako Quick Staining kit (Dako, Carpinteria, CA) enabled the shortest incubation times but other primary antibodies which required prolonged incubation times up to 10 rain also allowed identification of cells and successful collection of RNA. RNase inhibitor (200 to 400 U/ml) was added to the primary antibody and the color development steps. An altemative method for the identification of cells within a tissue section is to stain one section using a rapid staining protocol. The stained cells of interest in this section are then used as a guide to determine which cells should be captured from an adjacent section which has been stained using a rapid general stain (Fig. 1). This approach reduces the number of treatment steps before the RNA isolation which also reduces the risk of RNA degradation.

Cell Capture and Storage This review focuses on the techniques involved in capturing cells using the Arcturus Engineering PixCell II system (Mountain View, CA) but the methods outlined are equally applicable to the preparation of specimens and the isolation of RNA using other laser capture systems. Immediately prior to capturing the tissue sections on the slides are flattened using an electrostatic charged plastic film (PrepStrip Tissue preparation strips 9 j. Pail, E. J. Kunkel,U. Gosslar,N. Lazarus,P. Langdon,K. Broadwell,M. A. Vierra,M. C. Genovese, E. C. Butcher, and D. Soler, J. lmmunol. 165, 2943 (2000). l0 E Fend, M. R. Emmert-Buck,R. Chuaqui, K. Cole, J. Lee, L. A. Liotta, and M. Raffeld,Am. Jo Pathol. 154, 61 (1999).

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FIG. 1. Identification and capturing of airway smooth muscle cells. Five #m serial sections of human airway tissue were cut and stained with (a) mouse anti-human alpha smooth muscle actin and the Dako LSAB2 new fuchsin detection system and counterstained with Mayer's hematoxylin for the identificationof the different cell types or (b) rapid Mayer's hematoxylinand eosin and viewed through a visualizer on the LCM microscope (roadmap image). For capturing, the H&E stained section was viewed without the visualizer demonstratingthe (c) before image, (d) after image showing where the captured cells were removed, and (e) cap image of the captured cells. Arcturus) which also serves to remove cells only loosely attached to the slide surface. Cells of interest are captured onto either the standard L C M caps or the CapSure High Sensitivity L C M caps (HS). The standard caps bind more nonspecific material than the HS caps when certain tissue types are studied (for example, human lung and mouse brain). This nonspecific material must be removed using either the adhesive strip on the rear of a Post-It note (3M, St. Paul, MN) or the CapSure cleanup pads from Arcturus, although the adhesive force of the CapSure strips can remove some of the specific material if extreme care is not taken. Once the cells of interest have been captured, the caps are placed on the 0.5 ml tubes in the presence of 1 0 0 / z l lysis buffer for standard caps or 1 0 - 2 0 / z l for HS caps. Arcturus now provides ExtracSure sample extraction devices with the HS caps which enable the cell lysis buffer to be added to a much smaller area of the cap surface thereby reducing the volume required and enhancing the effective yield of RNA. In our experience Eppendorf (Hamburg, Germany) 0.5 ml micro

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test tubes fit the caps snugly but alternative brands may be appropriate. The tubes can be stored at -80* for at least 1 month without appreciable RNA degradation but should be kept inverted to ensure that the lysis buffer is in contact with the cells. RNA I s o l a t i o n RNA isolation from laser captured cells requires the same procedure as a standard RNA isolation but on a small scale. Many manufacturers are now producing kits for RNA isolation from small numbers of cells and, in our hands, the kits tested all gave reasonable yields for the number of cells processed. The column-based isolation procedures generally provide a greater yield than the methods that rely on multiple phenol-chloroform washes to clean the RNA, as a percentage of the RNA is lost at each wash step. The columns that have a smaller surface area, many of which are now associated with the RNA isolation kits for small cell numbers, enable the RNA to be eluted in a smaller volume, thereby reducing the need for concentrating the RNA by precipitation or vacuum centrifugation. Although it is still possible to develop a method for RNA isolation using individual reagents made up in the laboratory, the nature of the samples available and the time taken for LCM have resulted in many groups turning to kit-based isolations to avoid the loss of precious samples as the method is developed. R N A Isolation Kits

One of the earliest kits to be marketed for use with LCM samples was the Stratagene (La Jolla, CA) Micro RNA isolation kit. This kit has been reported by many users of LCM to be a successful method for RNA isolation from LCM captured cells) °-13 Some users have adapted this kit to adjust the phenol-chloroform wash steps. 7 More recently Stratagene has released the Total RNA Microprep kit which has been further optimized for isolation of RNA from LCM captured cells. In our hands this kit works well. This newer kit is a column-based extraction which does not contain the phenol-chloroform steps. The Qiagen (Valencia, CA) RNeasy mini kit is another column-based kit that has been reported to yield good quality RNA from LCM samples. 14 The widely used RNA isolation reagent from Life Technologies (Invitrogen, Carlsbad, CA), Trizol, reacted adversely with the thermoplastic film on the LCM caps and consequently is not appropriate for the isolation of RNA from LCM. 11L. Luo,R. C. Salunga, H. Guo, A. Bittner, K. Joy,J. E. Galindo, H. Xiao, K. E. Rogers,J. S. Wall, M. R. Jackson,and M. G. Erlander, Nat. Med. 5, 117 (1999). 12T. N. Darling, C. Yee,J. W. Bauer, H. Hintner, and K. B. Yancey,J. Clin. Invest. 11}3,1371 (1999). 13R. B. Nagle, J. Histochem. Cytochem. 49, 1063(2001). 14M, Neira, V. Danilova,G. Hellekant, and E. A. Azen,Mamm. Genome 12, 60 (2001).

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Arcturus has released its own version of the RNA isolation kit for use with the LCM caps. This kit is reported to have been optimized for use with the very small numbers of cells isolated during capturing, but to date there are no independent data evaluating the efficiency of this kit. Following RNA isolation, or as part of the isolation procedure, the sample needs to be treated with RNase-free DNase I to remove any contaminating DNA. In the Stratagene Total RNA Microprep kit the DNase treatment is included during the RNA isolation procedure. For other RNA isolation protocols, the DNase treatment is usually carried out after the RNA has been redissolved in DEPCwater by incubating with 20 U of DNase I for 1 to 2 hr at 37 °. The DNase I is removed with a phenol-chloroform wash followed by RNA precipitation in isopropanol in the presence of sodium acetate and glycogen carrier (10/zg//zl) or by purifying the RNA by binding it to a column and washing twice before eluting the RNA from the column. Appropriate columns include those used for RNA isolation or Microcon-100 columns (Millipore, Bedford, MA). Once again, the columns with the smaller surface area allow the RNA to be eluted in a smaller volume. Alternatively, if the downstream application is reverse transcriptase-polymerase chain reaction (RT-PCR) the primers can be designed to cross intron-exon boundaries to prevent the amplification of any contaminating genomic DNA.

RNA A n a l y s i s

Reverse Transcriptase-Polymerase Chain Reaction RT-PCR is often used to measure the gene expression for particular genes of interest in RNA isolated from LCM samples. In some cases the RNA lysis buffer is taken straight from the caps to the RT-PCR, but it is more usual to isolate the RNA completely before commencing downstream analysis. The RNA can be reverse transcribed to cDNA using random hexamers or oligo(dT)primers. Gene-specific primers can then be used for the PCR. This method will allow the examination of multiple genes from one reverse transcription reaction. Alternatively, the RT-PCR can be performed as a one-step reaction using the gene-specific primers for the reverse transcriptase reaction and the amplification reaction. Real-time PCR is being used where available for the detection of gene expression from LCM RNA samples, as the sensitivity of this technique requires less starting material than a standard PCR run to produce a reliable result. The method described here uses the Applied Biosystems equipment and reagents, but any of the real-time PCR systems can be used for this application. A real-time RT-PCR is prepared using the TaqMan One-Step RT-PCR Master Mix Reagents Kit (PE Applied Biosystems). For precise quantitative analysis of gene expression primers and probe for a control gene, for example the

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Pre-developed TaqMan Assay Reagents [Endogenous Control Ribosomal RNA Control (18s rRNA)] (PE Applied Biosystems), can be included in the RT-PCR reactions. Ten microliters of total RNA isolated from LCM captured cells is analyzed in a 25 #1 reaction containing 1x Master Mix, 1× MultiScribe and RNase Inhibitor Mix, 50-900 nM gene-specific forward primer, 50-900 nM gene-specific reverse primer, 50-250 nM gene-specific probe, and 1x control Primer and Probe Mix. The concentration of the gene-specific primers and probe needs to be optimized for each reaction. The multiplexing of the control gene and the gene-specific primers and probe also needs to be individually assessed. RT-PCR reactions are performed in the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycle conditions consist of reverse transcription at 48 ° for 30 min, denaturation at 95 ° for 10 min, followed by 40 cycles of 95 ° for 15 sec and 60 ° for 1 min. Data from the reaction are collected and analyzed by the complementary computer software. Reverse transcription can also be performed with random hexamers or oligo(dT)primers as described previously and the real-time PCR performed using the TaqMan Universal PCR Master Mix (PE Applied Biosystems). SYBR Green detection methods using the appropriate master mix can also be used for the detection of the amplified product with a melting curve analysis ensuring the specificity of the amplification. The sensitivity of this detection technique allows the reliable detection of sequences from as few as 100 cells (Fig. 2). The number of cells required for detection varies with cell type as different cell types contain different amounts of RNA. Where available, cells grown in culture can be used to estimate the number of cells required for the detection of specific genes. Adherent cells can be grown directly on the microscope slides before fixation and staining for LCM. Precise cell numbers captured are easier to calculate using this method but caution should be exercised in equating cell numbers captured from tissue culture cells to cell numbers captured from tissue sections, as the cells cultured on the slides are not subjected to sectioning and are therefore whole cells. The cells isolated from sections are not all whole, so consequently the RNA yield is lower. In our experience, there is a difference of about 100-fold in the RNA yield for smooth muscle cells. cDNA Microarrays

RNA isolated from LCM captured cells can also be applied to cDNA or oligonucleotide microarrays. Before labeling for array analysis, the mRNA is amplified linearly11,15 to increase the amount of sample but still reflect the proportions of message for each gene in the original sample.

15j. Eberwine, H. Yeh, K. Miyashiro,Y. Cao, S. Nair, R. Finnell, M. Zettel, and P. Coleman,Proc. Natl. Acad. Sci. U.S.A. $973010 (1992).

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267

40 35 30 25 20 15 10 5 0

i

i

100~ RNA

1000

100

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FIG.2. Real-time RT-PCR following LCM. Human airway smooth muscle ceils were grown on microscope slides before 70% ethanol fixation and rapid H&E staining. Ten, 100, or 1000 cells were captured on HS caps (n = 6) and the RNA isolated using the Stratagene Total RNA Microprep kit. Real-time RT-PCR was used to quantify the expression of a control and target gene using a multiplex RT-PCR with two dyes. One hundred nanograms of RNA isolated from human cultured airway smooth muscle cells was used as a positive control and the absence of RNA template (NTC) as the negative control.

Linear Amplification. The R N A is isolated as described above and, after DNase I treatment, resuspended in 11/zl DEPC-water. One microliter of R N A is kept as the negative control for real-time RT-PCR. Reverse Transcription. The remaining 10/zl is mixed with 1/zl 0.5 mg/ml T7oligo(dT) primer ( 5 t T C T A G T C G A C G G C C A G T G A A T T G T A A T A C G A C T C A C TATAGGGCGT21 - 3 ' ) 11 and heated to 70 ° for 10 min. The sample is immediately placed on ice while 4 / z l first strand reaction buffer, 2 / z l 0.1 M DTF, 1/zl RNasin and 1 / z l 10 m M dNTPs [Life Technologies (Invitrogen), Carlsbad, CA] are added. Incubation at 42 ° for 5 min is carried out before the addition of 1 / z l Superscript II (Life Technologies) and the continuation of the incubation at 42 ° for 1 hr. To the sample is added a cocktail containing 9 2 / z l water, 3 0 / z l 5x secondstrand synthesis buffer, 3 #1 10 m M dNTPs, 4 / z l D N A polymerase I, 1/zl RNase H, and 1 #1 Escherichia coli D N A ligase and this mix is incubated at 16 ° for 2 hr. Two microliters of T4 D N A polymerase is then added and the sample incubated at 16 ° for a further 15 min. The enzymes are then inactivated by heating to 70 ° for 10 min and the c D N A extracted by the addition of 150 #1 p h e n o l - c h l o r o f o r m and spinning at 14,000 rpm for 5 rain. The aqueous phase is washed 3 times with 5 0 0 / z l water in a Microcon-100 column (Millipore) and eluted in 8 / z l of water. In Vitro Transcription. Using the Ampliscribe T7 transcription kit (Epicentre Technologies, Madison, WI) a cocktail of 2 / z l 10x Ampliscribe T7 buffer, 1.5/zl

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each of 100 mM ATE UTE CTP, and GTP, 2/zl 0.1 M DTT, and 2/zl T7 RNA polymerase is added to the cDNA. All reagents except the enzymes should be at room temperature before the cocktail is mixed. The mixture is incubated at 42 ° for 3 hr before the addition of 1 #1 DNase I and a further 20 min at 37 °. The amplified RNA (aRNA) is then washed three times using a Microcon-100 column and eluted in 11/zl water. One microliter is kept for analysis by real-time RT-PCR to check the efficiency of the amplification and the remaining 10 #1 is carried on for a second round of amplification. Second Round Amplification. To the 10/zl aRNA is added 1/zl random hexamers (1/zg/#l) and the sample is incubated at 70 ° for 10 rain before bein~ chilled on ice and equilibrated to room temperature. To this is added 4/zl 5x first-strand buffer, 2/~10.1 M DTT, 1 #1 10 mM dNTPs, and 1/zl RNasin. After 5 min at room temperature 1/zl Superscript II is added. After a further 5 min at room temperature the mixture is incubated at 37 ° for 2 hr. One microliter of RNase H is then added and the incubation continued at 37 ° for a further 20 rain before inactivating the enzyme at 95 ° for 2 rain and placing on ice. One microliter of 0.5 mg/mi T7 oligo(dT)primer is added and the mixture incubated at 70 ° for 5 rain followed by 42 ° for 10 min. To this is added 90/zl water, 30/zl 5x second-strand synthesis buffer, 3/zl 10 mM dNTPs, 4 #1 DNA polymerase I, and 1 #1 RNase H and the mixture is incubated at 16 ° for 2 hr. Two microliters T4 DNA polymerase is added and the incubation is continued at 16 ° for a further 10 min. The sample is then heated to 65 ° for 10 rain before being extracted with 150/zl phenol-chloroform and purification using the Microcon-100 columns. The cDNA is then put through a second round of T7 in vitro transcription as described above (Fig. 3) before a final purification ready for labeling using a standard microarray labeling protocol. After the final round of amplification, 1/zl of the aRNA is taken for real-time RT-PCR to check the efficiency of the amplification reaction. Following the linear amplification of the mRNA, the aRNA can be labeled using a standard microarray labeling reaction before hybridization with the microarray using a standard protocol for a microarray experiment. It has been reported that between 20013 and 100011 cells are enough for RNA linear amplification and subsequent microarray analysis. DNA Analysis DNA Isolation DNA can be isolated from fresh frozen tissue samples but can also be isolated from paraffin embedded samples providing a much wider set of source material for studies of genes using DNA-based techniques. Ten micron tissue sections are cut on the cryostat and mounted on clean, flat microscope slides. Sections are stained appropriately for the identification of the cells types being studied. Hematoxylin and eosin are commonly used. Ceils are captured onto the standard or HS LCM caps and the caps stored in a manner similar to that described for RNA isolation. Once again DNA isolation is carried out using scaled-down standard isolation

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269

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FIG.3. Linear amplificationof RNA. Schematic of the amplificationmethodused for increasing the amountof mRNAavailablefor downstreamanalysis,aRNA, AmplifiedRNA. methods. Twenty to 50/~1 of extraction buffer containing 10 mM Tris pH 8.0, 2 raM EDTA, 0.2% Tween 20, and 200/zg/ml proteinase K is incubated with the cells on the cap (in an inverted 0.5-ml Eppendorf tube) at 37 ° overnight. The following day the mixture is heated to 100 ° for l0 rnin to inactivate the proteinase K and 3 to 5% of the resultant solution can be used directly as PCR template. The QIAamp tissue kit (Qiagen, Valencia, CA) has been reported to produce quality DNA.

Loss of Heterozygosity and Mutation Analysis Standard PCR conditions using primers designed to flank mutation sites can be used to detect mutations in LCM isolated samples. One or two rounds of PCR

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amplification consisting of 35 to 40 cycles followed by direct sequencing has been used to identify FAS gene mutations in prostatic intraepithelial neoplasia and concurrent carcinoma, 16 androgen-regulated homeobox gene NKX3.1 mutations in benign and malignant prostate epithelium, 17 and ICl-ras and/or p53 mutations in tumor genotyping) 8 Takayama et al. also used DNA amplification to look for loss of heterozygosity at four known polymorphism sites in prostatic intraepithelial neoplasia. 16 Methylation Analysis Patel et al. 19 have described a method for the analysis of the methylation state of a gene (p16 Ink4a) DNA isolated from LCM captured cells. Briefly, the DNA is isolated from the cells by the addition of 50/zl lysis buffer (0.5% Tween 20, 1 mM EDTA pH 8.0, 50/zM Tris pH 8.5, and 0.5/zg//zl proteinase K) and incubation overnight, inverted, at 37 °. The samples are then incubated at 95 ° for 8 min before precipitation of the DNA by the addition of 1.8 ml 100% ethanol in the presence of 2 / z l glycogen (20 mg/ml). The DNA is washed in 70% ethanol twice and resuspended in 10/zl water. The samples are digested overnight at 37 ° with E¢oRI in a total volume of 20/zl before denaturation at 75 ° with 2/zl 3 M NaOH. Bisulfite modification is then carried out by the addition of 250 #14.8 M sodium bisulfite and 14/x120 mM hydroquinine before overlaying the samples with light mineral oil and incubating at 55 ° for 5 hr. The DNA is purified using Centricon-30 columns (Millipore) and eluted in 100/zl water. The samples are then desulfonated with 4.5/zl 3 M NaOH and neutralized with 28/~1 5 M ammonium acetate. The DNA is precipitated with 3 volumes of 100% ethanol, in the presence of 1/zg glycogen, overnight at - 2 0 ° and then washed with 70% ethanol and resuspended in 20/zl water. Two rounds of nested PCR can then be performed using primers that have been designed with all of the cytosines replaced with thymines to enable the amplification of the bisulfite-treated DNA. The unmethylated cytosines are converted to uracils during the modification with sodium bisulfite. During the subsequent PCR the uracils are converted to thymine residues. This method allows site-specific and region-specific methylation to be studied) 9 16 H. Takayama, T. Takakuwa, Z. Dong, N. Nonomura, A. Okuyama, S. Nagata, and K. Aozasa, Lab, Invest. 81, 283 (2001). 17 D. K. Ornstein, M. Cinquanta, S. Weiler, E H. Duray, M. R. Emmert-Buck, C. D. Vocke, W. M. Linehan, and J. A. Ferretti, J. Urol. 165, 1329 (2001). 18 D. Dillon, K. Zheng, and J. Costa, Exp. Mol. Pathol. 70, 195 (2001). 19 A. C. Patel, C. H. Anna, J. E Foley, E S. Stockton, E L. Tyson, J. C. Barrett, and T. R. Devereux, Carcinogenesis 21, 1691 (2000).

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[23] Analysis of Specific Gene Expression By GEORGIALAHR, ANNA STARZINSKI-POWITZ,and ANETrE MAYER Introduction One of the key points for understanding the molecular basis of development or carcinogenesis is the analysis of gene expression in specific cell populations. To isolate these cell populations laser-assisted microdissection is commonly used. Here, the P.A.L.M. Robot-MicroBeam system allows contamination-free isolation of single cells or defined cell clusters from frozen or archival tissue sectionsJ After laser microbeam microdissection (LMM) cells of interest are catapulted by the force of the laser directly into the cap of a common microfuge tube. This catapulting process is called "laser pressure catapulting" (LPC).I,2 Microdissected cells are now suitable for subsequent experiments. Many different methods for RNA isolation and RT-PCR analysis of cells from cryopreserved and archival tissues have been published in the past few years. 3-7 The decision to use paraffin-embedded or frozen tissue depends on subsequent experiments and tissue supply. Paraffin-embedded tissues may be used for retrospective studies and may permit tracking over long periods of time. However, cross-linking of proteins with nucleic acids by formalin, which is the most common fixative, leads to RNA or DNA fragmentation after nucleic acid isolation procedures, making them useless for some downstream methods. Because of these strand breaks the length of an amplifiable RT-PCR product from archival tissue should not exceed 380 bp. For isolation of longer fragments and of nearly undegraded RNA (e.g., for library construction or array technique) cryopreserved tissue should be chosen since the quality of the isolated RNA is much higher. Cryopreserved tissue sections require some changes in protocol (see below) since mild fixatives, which keep RNA and DNA almost undegraded, are not able to completely inhibit endogenous RNase activity. Starting with archival or cryopreserved laser-microdissected cells, we established improved protocols for the isolation of RNA and downstream RT-PCR analysis, including Real-time RT-PCR. The combined LMM and LPC provide 1 K. Schtitze and G. Lahr, Nat. Biotechnol. 16, 737 (1998). 2 K. Schtitze, H. P6sl, and G. Lahr, Mol. Cell Biol. 44, 735 (1998). 3 M. R. Bernsen, H. B. Dijkman, E. de Vries, C. G. Vigdor, D. J. Ruiter, G. J. Adema, and G. N. E Muijen, Lab. Invest. 78, 1267 (1998). 4 L. Jin, C. A. Thompson, X. Qian, S. J. Kuecker, E. Kulig, and R. V. Lloyd, Lab. Invest. 79, 511 (1999). 5 G. Lahr, Lab. Invest. 80, 1477 (2000). 6 G. Lahr, M. Stich, K. Schfitze, E Bliimel, H. Pt~sl, and W. B. J. Nathrath, Pathobiology 68, 218 (2000). 7 y. Imamichi, G. Lahr, and D. Wedlich, Dev. Genes Evol. 211, 361 (2001).



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an extremely useful tool for selecting and pooling morphologically similar ceils, which opens the field for the analysis of specific gene expression or differential gene expression screens between microarrays. S t e p I: C e l l / S p e c i m e n P r e p a r a t i o n

Buffers, Reagents, and Equipment 1. For tissue preparation: 1.35/zm thin polyethylene-naphthalene membrane (PEN; P.A.L.M. Microlaser Technologies AG, Bemried, Germany) 0.1% poly-L-lysine, Mayer's hematoxylin, eosin, and mineral oil (all Sigma, Deisenhofen, Germany) 2. For laser microbeam microdissection (LMM) and laser pressure catapulting (LPC): Robot-MicroBeam (P.A.L.M. Microlaser Technologies AG, Bernried, Germany) coupled onto an inverted Axiovert 135 microscope (Carl Zeiss, Gtttingen, Germany)

Procedure Specimen Preparation Formalin-preserved tissues. We commonly use 5/~m serial sections of a routinely formalin-fixed and paraffin-embedded tissue block obtained from the pathology department. 1. Mount the tissue sections either directly onto a common glass slide or onto a membrane-mounted glass slide. 2. For membrane mounting: use a droplet of 100% ethanol to attach the 1.35/zm thin PEN membrane to the glass slide. 3. Fix the edges of the membrane with nail polish. 4. Cover the nail polish after drying with an autoclave tape to avoid its dissolving during deparaffinization in xylene. 5. Cover the membrane with 0.1% poly-L-lysine prior to tissue mounting and air dry the coated membrane. 6. Mount the tissue section on the membrane, deparaffinize, and stain the sections with hematoxylin-eosin as usual.

Cryopreserved tissues. We prepare 8/zm serial sections of snap-frozen tissue derived from surgery on a cryostat. 1. Mount the PEN membrane on a glass slide (see points 2-4 above). 2. UV irradiation of the membrane for 20 min renders the hydrophobic membrane more hydrophilic.

[9-3]


3. 4. 5. 6.

273

Mount the frozen tissue section on the membrane. Fix the sections with 70% ethanol for 30 sec and rinse briefly twice in PBS. Stain sections with hematoxylin for 10 sec and rinse again twice in PBS. Dry sections by incubation for 15 sec each in 70%, 90%, and 100% ethanol.

Laser Microbeam Microdissection and Laser Pressure Catapulting 7. Insert the object slide onto the microscope stage (Figs. 1A, 2A, 2E, 3A). 8. Moisten the cap of a microfuge tube with a 2 #1 droplet of mineral oil by using a micropipette tip, or apply a 5 / z l droplet of lysis buffer (included in PureScript isolation kit) to the cap.

FIG. 1. Isolation and RT-PCR analysis of microdissected archival cells. Microscopic illustration of one specific experiment using LMM and LPC to capture pooled single cells from membrane-mounted archival hematoxylin-eosin stained tissue slices of a differentiated colon adenocarcinoma. The view shows specimen before LMM (A), the remaining tissue after LMM (B), the remaining tissue or cells after LPC (C), and the catapulted and captured ceils in the cap of a conventional microfuge tube (D). The experiment was performed by using a 40x objective. Panel E shows the MspI digests of Ki-ras2 RT-PCR-amplified codon 12 products (217 bp RT-PCR fragments) which were separated on a 1.5% ethidium bromide-stained agarose gel. The uncut RT-PCR fragment of 7 pooled tumor cells (shown in D) is indicated by (1). The MspIdigest of this sample is shown in 2. An RT-PCR sample of about 100 isolated SW480 cells digested with MspIwas loaded in lane S. A 100 bp ladder size marker (M) was run in parallel. The sizes of the 100 bp ladder are indicated on the right side as are the uncut 217 bp RT-PCR fragments (white arrow).

274

GENETICAPPLICATIONS

[23]

FIG. 2. Isolation and RT-PCR analysis of microdissected cryopreserved cells. Microscopic illustration of two specific experiments using LMM and LPC to capture epithelial (A-D) or stromal (E-H) cells from membrane-mounted cryopreserved hematoxylin-stained tissue sections of an endometriotic lesion. The view shows a specimen before LMM (A and E), the tissue after LMM (B and F), the remaining tissue after LPC (C and G), and the catapulted cells captured in the cap of a conventional microfuge tube (D and H; all 40x objective). (I) GP130RT-PCR analysis of microdissected epithelial (1) or stromal (2) cells derived from an endometriotic lesion. Second PCR products (220 bp) were loaded on 1.5% ethidium bromide-agarose gels. C1 is a H20 control of 1st PCR used as a template in 2nd PCR; C2 is control containing H20 instead of DNA. Long ~-actin fragments derived from about 2000 dissected endometrial cells are shown in (J), where (3) is from from 1st and (4) is from 2nd (nested) PCR. The sizes of the 100 bp ladder are indicated on the right side. The arrowheads indicate the faint signal of the 1st and the arrows correspond to the 2nd PCR products. M, 100 bp ladder size marker.

[23]

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Temperature (:C) FIG.3. Isolation,RT, and real-timePCR analysisof microdissectedarchivalcells. In this example LMM and LPC were used to capture a cell cluster of about 100 cells from an archivalhematoxylinstained tissue slice of a follicularvariantof a papillarythyroid carcinoma.The view shows specimen before (A) and after microdissectionand LPC (B), and the catapulted cells (arrowhead) in the cap (C; all 40x objective). The melting curve analysisafter 50 cycles in a real-timePCR reaction from RET/PTC1 LightCyclerSYBRGreen I assay is shownin (D). Here, the first negativederivativeof the fluorescence(-dF/dT) is plotted as a functionof temperature (°C; Tin).The RET/FTC1 meltingcurve analysisof the dissectatescomposedof about 100 laser-microdissectedcells is shownin black (1). The dotted line (T) indicatesthe translocation-positivecontrol,derivedfrom the tumorcell line TPC-1. 9. Insert the "prepared" cap into the LPC collector. 10. Focus the laser microbeam through a 20x or 40x dry objective lens. Energy settings are dependent on the absorption behavior of the specimen and on the transmission rate of the objective lens. 11. For microdissection adjust the laser energy either to solely cut the tissue (glass-mounted; Fig. 2B) or to cut the entire membrane-tissue stack (membranemounted; Figs. 1B, 3B, 3F). 12. For LPC adjust the energy settings. They should be sufficiently high to catapult the specimen into the microfuge cap, which is now centered above the line of laser fire by a special LPC-collector device. 13. Inspect the catapulted cells within the cap by using the "checkpoint" position of the system (Figs. 1D, 2D, 2H, 3C). 14. Remove the cap including the captured cells from the collector device.

276

GENETICAPPLICATIONS

[231

15. Cover the catapulted cells by a total volume of 30/zl of RNA lysis buffer. 16. Top the caps with the remaining tube and process as described below.

Notes to Step I Mounting of the PEN membrane takes less than 1 min per slide. The membranecovered slides can be stored at room temperature until needed. S t e p II: RNA I s o l a t i o n

Buffers, Reagents, and Equipment Enzymes and buffers are derived from the PUREscript RNA Isolation Kit. PUREscript RNA Isolation Kit (BIOzym Diagnostik, Hess. Oldendorf, Germany) Glycogen (MB grade; 20 #g//zl) (Roche Diagnostics GmbH, Mannheim, Germany)

Procedure Preparation of Total RNA from Microdissected Cell Samples Cell lysis 1. Add 30/zl Cell Lysis Solution to the catapulted cells in the cap and pipette up and down 15-30 times to lyse the cells. 2. Centrifuge at 13,000-16,000g for 1 min to pellet the lysis solution including the cells.

Protein-DNA precipitation 3. Add 10/zl Protein-DNA Precipitation Solution to the cell lysate. 4. Invert tube gently 10 times and place tube into an ice bath or cryo pack for 5 min. 5. Centrifuge at 13,000-16,000g for 3 minutes. The precipitated proteins and DNA will form a tight white pellet.

RNA precipitation 6. Pour the supernatant containing the RNA (leaving behind the precipitated protein-DNA pellet) into a clean microfuge tube containing 30/zl 100% 2-propanol. 7. Add 1.5/zl of glycogen (1 : 10 diluted with H20) as a carrier. 8. Mix the sample by inverting gently 50 times. Optional: incubate at 4 ° for 20 min. 9. Centrifuge at 13,000-16,000g for 5 min; the RNA will be visible as a small, translucent pellet.

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277

10. Pour off the supernatant and drain tube briefly on clean absorbent paper. Add 30 #1 70% ethanol and invert the tube several times to wash the RNA pellet. 11. Centrifuge at 13,000-16,000g for 3 min. Carefully pour off the ethanol. 12. Invert and drain the tube on clean absorbent paper and allow sample to air dry 10-15 min or for a maximum of 5 min in a Speed Vac.

RNA hydration 13. Add 5 #1 RNA Hydration Solution. 14. Allow RNA to rehydrate for at least 30 min on ice. Alternatively, store RNA sample at - 7 0 ° to - 8 0 ° until use. 15. Before use vortex sample vigorously for 5 sec and pulse spin. 16. Store purified RNA sample at - 7 0 ° to - 8 0 ° or proceed with cDNA synthesis.

Notes to Step H Longer incubation and centrifugation steps may increase total RNA yield. Chemicals and staining dyes, such as hematoxylin and eosin, and dyes from an optional immunostaining procedure may inhibit the downstream RT-reaction, but this effect is negated by purification of the "cell extracts" by RNA isolation. 5 S t e p III: R e v e r s e T r a n s c r i p t i o n

Buffers, Reagents, and Equipment ExpeRT-PCR Kit (Hybaid-AGS, Heidelberg, Germany) dNTP solution containing all four dNTPs (10 mM each) RNase inhibitor (40 units//zl) (Hybaid-AGS, Heidelberg, Germany) Hexanucleotide Mix, 10× concentration (random hexamers; Roche Diagnostics GmbH, Mannheim, Germany)

Procedure Preparation of cDNA 1. RNA must be denatured for 5 min at 80° and immediately transferred to ice before being added to the reaction mix. 2. Set up the RT reaction mix in a total volume of 12.5/zl: 10x ExpeRT-PCR-Buffer, complete (including 1.75 mM MgC12 final concentration) DMSO (2% final concentration) Template: total RNA 10x Random hexamers

1.25/zl 0.25/zl 5/zl 0.75/zl

278

GENETICAPPLICATIONS 10 mM dNTP-Mix 40 units//zl RNase inhibitor H20, double distilled, DEPC treated 5 units/#l AMV Reverse Transcriptase

[231 0.25/zl 0.06 #1 4.7/zl 0.25/zl

3. Incubate 60 min at 42 °. 4. Inactivate AMV Reverse Transcriptase (10 minutes at 80°).

Notes to Step III a. To reduce the chance of contamination with exogenous nucleic acids, prepare and use a special set of reagents and solutions for RNA isolation, RT and PCR only. b. Temperature for RT: any template denaturation to overcome secondary structures in the RNA should be performed in the absence of the AMV-Reverse Transcriptase, as the enzyme is denatured at elevated temperatures. AMV-Reverse Transcriptase has optimum activity at 42 ° [using oligo(dT) primers and random hexamers] but can be used at temperatures of up to 60 ° (using gene-specific primers) to minimize problems with secondary structures. c. RNase inhibitor: the use of RNase inhibitor is optional.

S t e p IV: P C R A n a l y s i s

Buffers, Reagents, and Equipment 1. For PCR in the Thermal Block Cycler: ExpeRT-PCR Kit (Hybaid-AGS, Heidelberg, Germany) Thermal block cycler instrument 9600 (PerkinElmer Applied Biosystems GmbH, Weiterstadt, Germany) 2. For real-time PCR in the LightCycler: LightCycler instrument (Roche Diagnostics GmbH, Mannheim, Germany) LightCycler capillaries (Roche Diagnostics GmbH) LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH) 3. Special equipment: Barrier tips for micropipettes Microfuge tubes (0.5 ml, thin-walled for PCR amplification) Thermal block cycler programmed with desired amplification protocol Water baths or heating blocks, preset to 80 ° and to 42 ° Ice-water bath or cryopack 1.5% Agarose gels in TAE buffer containing 0.25/zg/ml ethidium bromide TAE buffer: 40 mM sodium acetate (pH 8.3), 40 mM Tris base, 2 mM EDTA

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279

Procedure A. PCR Reactions in Block Cycler 1. Pipette the following PCR reaction mix (1 st or 2nd PCR) in a total volume of 25/zl: 10× ExpeRT-PCR-Buffer (complete) (including 1.75 mM MgC12 final concentration) Template: aliquot of RT- or 1st PCR reaction 10 pmol/~l 5'-Primer 10 pmol//zl 3'-Primer 10 mM dNTP-Mix 2.5 units/#l Proof Sprinter Taq/Pwo Mix DMSO (2% final concentration); note: 2% DMSO is already included in the RT- and 1st PCR mix MgC12 (1.75-5 mM MgC12 final concentration) H20 double distilled (adjust to a total volume of 25 #1)

2.5 #1 3.1-10 #1 1 #1 1 #1 0.5/zl 0.2 #1 x/zl x #1 x/zl

2. Subject the samples to 1 cycle at 94 ° for 2 min, 35-45 cycles at 93 ° for 30 sec, at 56-62 ° for 30 sec (this is dependent on the primer composition and on the amount of template DNA), at 72 ° for 1 min; at 72 ° for 10 min, and storage at 4 °. 3. For detection of PCR products analyze the sizes of the amplified products of the 1st and 2nd PCR reaction, using a 15-20/zl aliquot of each reaction, by agarose gel electrophoresis. Notes to Step IV, A a. Sense and antisense primers (10/zM each) in H20. Each primer should be 20-25 nucleotides in length and have a GC content of approximately 50-60%. b. Oligonucleotide primers synthesized on an automated DNA synthesizer can generally be used in RT-PCR without further purification. c. Magnesium is required by both the AMV and the Taq/Pwo mix and should be optimized for each PCR reaction. d. Times and temperatures may need to be adapted to suit other types of equipment and reaction volumes. e. If the thermal cycler is not equipped with a heated lid, use either mineral oil or paraffin wax to prevent evaporation of liquid from the reaction mixture during RT and PCR. f. Controls containing H20 instead of DNA (negative) and containing cDNA or DNA (positive), e.g., from a cell line, are always run in parallel. The results of a specific RT-PCR analysis using archival microdissected cells from a colon adenocarcinoma are shown in Fig. 1E. Related single and oligo cell

280


[231

analyses have been published.l'5 Briefly, after microdissection and RT-PCR analysis one aliquot of the 2nd Ki-ras2 PCR reaction, example shown for the seven pooled tumor cells in Figs. 1A-1D and for 100 pooled immunostained SW480 cells, was digested with the restriction enzyme MspI. The SW480 tumor cell line differs from the wild-type Ki-ras2 at codon 12. 8,9 MspI cuts within the 217 PCR fragment only if codon 12 is wild type) ° The products of the nested PCR and the MspI digests were fractionated on 1.5% agarose-ethidium bromide gels to yield the "uncut" DNA fragments of the appropriate length (Fig. IE). This indicates that both the microdissected tumor cells and the SW480 cells carry a mutation in codon 12 of the Ki-ras2 gene. The results of a specific RT-PCR analysis using microdissected cells from cryosections of endometriotic lesions are shown in Figs. 2I-2J. Tissue-specific expression of GP130 mRNA was investigated by first microdissecting either epithelial (Figs. 2A-2D) or stromal (Figs. 2E-2H) cells. RT-PCR analysis of 100300 cells detects GP130 mRNA in both cell populations; aliquots of the second GP130 PCR reaction are examples shown in Fig. 2I. The use of cryosections allows the isolation of large RNA fragments; this is demonstrated by the amplification of a 1.7 kb (first PCR) and a 755 bp (second PCR) fl-actin fragment (Fig. 2J). B. Real-Time PCR Used for Archival Tissue 1. Pipette 60/zl from the LightCycler-FastStart Reaction Mix SYBR Green I into the LightCycler-FastStart Enzyme ("HotStart" reaction mix), mix gently, and protect from light. 2. Pipette the LightCycler-Sybr Green I PCR reaction mix (1st or 2rid PCR) in a total volume of 20/zl. "HotStart" reaction mix Template: RT- or 1st PCR reaction product 10 pmol//zl Y-Palmer 10 pmol//zl 3'- Primer 25 mM MgC12 (3 mM final concentration) H20 double distilled (adjust at a total volume of 25/zl)

2 #1 6.5/zl or 3/zl 1 #1 1/zl 1.6/zl x/zl

3. The amplification is performed in the LightCycler running 50 cycles for 15 sec at 95 °, 5 sec at 56-60 ° (depending on the oligonucleotides), and 10 sec at 72 °, starting with a 10 min denaturation at 95 °. 8W. Jiang, S. M. Kahn, J. G. GuiUen,S.-H. Lu, and B. Weinstein, Oncogene4, 923 (1989). 9 M. Verlann-deVries, M. E. Bogaard,H. van den Elst, J. H. van Boom, A. J. van der Eb, and J. L. Bos, Gene 50, 313 (1986). 10A. Haliassos,J. C. Chomel,S. Grandjouan,J. Kruh,J. C. Kaplan,and A. Kitzis,NucleicAcids Res. 17, 8093 (1989).

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4. For detection of PCR products assess the specificity of the amplified PCR products by performing melting curve analysis with the LightCycler software. Melting analysis was performed for 0 sec denaturation at 95 °, hybridization for 15 sec at 65 ° (depending on the target sequence), and continuously increasing the temperature (0.1 °/sec) from 65 ° (depending on the target sequence) to 95 °. 5. If desired, analyze the sizes of the amplified products of the PCR reaction in a 15-20/zl aliquot of each of the reactions by agarose gel electrophoresis. Notes to Step IV,, B a. SYBR Green I is a dye, specific for double-stranded DNA. It can be used for the amplification on every DNA or cDNA target. Each protocol needs adaptation to the appropriate reaction conditions. b. The amplicon size should not exceed 1 kb in length. For optimal results, select a product length of 700 bp. c. Use primers at a final concentration of 0.3-1 /zM each. A recommended starting concentration is 0.5/zM each. d. For specific and efficient amplification using the LightCycler instrument, it is essential to optimize the target-specific MgCI2 concentration that may vary from 1 to 5 mM. Real-time RT-PCR analysis has become an indispensable tool for rapid testing of various biological specimens, such as blood and biopsies, for the presence of gene mutations, translocations, and microorganisms. The results of a real-time RT-PCR to analyze a specific translocation using archival microdissected cells from a follicular variant of a papillary thyroid carcinoma are shown in Fig. 3D. There, the tumor tissue section was mounted directly onto a conventional glass slide. After microdissection and RT one aliquot was applied to real-time PCR. The melting curve analysis after 50 cycles from the RET/PTC 1 assay (see reference 6) is shown in Fig. 3D, where the first negative derivative of the fluorescence (-dF/dT) was plotted as a function of temperature (°; Tin). Analysis of about 100 lasermicrodissected cells resulted in a melting peak at about 87 °, which corresponds to the expected melting peak derived from the tumor cell line TPC-1 (T) used as a RET/PTC 1 translocation-positive control. Acknowledgments The authors thank M. Sfich and K. Schiitze for technical assistance and instrumental support. This work was supported by a grant of the Boehringer Ingelheim Sfiftung to A.S.-P.

282

GENETIC

(241 Gene Discovery By

MAUFWIO

1241

APPLICATIONS

with Laser Capture Microscopy NEIRA

and EDWIN

AZEN

Introduction The isolation of pure cell populations, suitable for nucleic acid extraction, from complex tissues by laser capture microdissection (LCM) is the basis for the straightforward method of gene discovery presented in this article. We describe, in detail, a fast method for the construction of cDNAs from material obtained by LCM and an example leading to the finding of new genes specifically expressed in taste cells as opposed to the surrounding epithelium. Taste buds are onion-shaped specialized neuroepithelial cell structures of 50-100 cells, 50 pm in size and commonly found embedded in tongue taste papillae. These characteristics make taste buds suitable for microdissection using a 30- to 50-pm laser beam. A detailed procedure will be given for the method we used to make cDNAs from material obtained by laser capture microdissection, and for all other procedures involved in the gene discovery process we will point out the relevant stages and will refer the reader to the specific kit or source describing the detailed procedure. General

Considerations

Laser capture microdissection performed on frozen sections of rhesus monkey circumvallate taste papillae (easily visible in the back of the tongue) was used to obtain two separate populations of cells: taste buds and epithelial cells immediately surrounding the taste buds.’ The differential screening strategy for the discovery of genes, specifically expressed in taste buds, is as follows: 1. Construction of a ;1ZAP II cDNA library from the microdissected taste buds 2. Plating of a portion of the taste bud library in duplicate filters 3. Hybridization with radioactively labeled complex cDNA probes obtained from the microdissected taste buds and microdissected surrounding epithelium 4. Selection of clones giving a positive signal with the taste bud cDNA probe but not with the control probe 5. Selection of clones not showing signals with either probe, as they may represent low-abundance genes missed out in the differential screening due to sensitivity ’ M. Neira, V. Danilova,

G. Hellekant,

and E. Azen, Mammalian

Genome

12,60

Copyright METHODS

IN ENZYMOLOGY,

VOL. 356

(2001).

2002, Elsevier Science (USA). Au rights reserved. 0076~6879102 $35.00

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WITH

LCM

283

issues involved in using complex cDNAs as probes. In order to determine to what cell population they are specific to, a number of clones from this set were chosen randomly, sequenced, PCR,primers designed, and PCR analysis with specific primers performed on separate cDNAs from taste buds and from surrounding epithelium. Clones showing a PCR signal in the taste bud cDNA population but not in the cDNAs from surrounding epithelium are selected as potentially taste bud specific. 6. Final selection of clones as taste cell specific is made by RNA in situ hybridization. Reagents The following reagents and buffers are necessary for the cDNA synthesis and are purchased from different companies or prepared as follows: 1. Dynabeads mRNA purification kit from Dynal: contains oligo(dT)25 beads, 2x binding buffer, washing buffer, and magnetic stand 2. Superscript RT II from GIBCO: contains reverse transcriptase, 0.1 M DTT, 5 x first-strand buffer 3. RNase inhibitor from GIBCO 4. T4 DNA polymerase and 10x T4 buffer from Epicentre 5. RNase H and RNase H buffer from Boehtinger Mannheim 6. Terminal deoxynucleotidyltransferase (TdT) and 10x One-Phor All plus buffer, from Pharmacia 7. Tuq polymerase, dATP, and dNTPs from Epicentre 8. 1 M TMAC from Sigma (a chemical that causes an oligonucleotide to hybridize based on length and not GC content which results in an improvement in the quality of the PCR product2) Preparation of different reaction mixtures is as follows: 1. 2x first-strand buffer: from 5x first-strand buffer with RNase-free water 2. Reverse transcriptase mix: 5 x First strand buffer RNase-free water 0.1 M DTT 10 mM Each dNTPs mix RNase inhibitor (10 UIpl) * K. N. Lambert

and V. M. Williamson,

Methods

44 11 CL1 2 I*1 1 Pl 1 Pl Mol. Biol. 69,l

(1997).

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[24]

3. T4 DNA polymerase reaction mix: Double distilled H20 10x T4 buffer 10 mM Each dNTPs Mix T4 polymerase (1 U//zl)

41.5/zl 5.0/zl 2.5/zl 1.0/zl

4. RNase H buffer: 20 mM Tris-HCl pH 8.0, 50 mM KC1, 10 mM MgC12, 1 mM DTT (prepare fresh) 5. RNase H reaction mix: RNase H buffer RNase H (1 U//zl)

20/zl 0.5/zl

6. Terminal transferase mix: Double distilled H20 10x One-Phor All plus buffer 1.5 mM dATP TdT (22 U//zl)

14/zl 2/zl 3/zl 1/zl

7. 5 x PCR buffer: 100 mM Tris-HC1 pH 8.3, 12.5 mM MgClz, 125 mM KC1, 0.25% Tween 20. 8. Primers: Chosen from Ref. 3. AL1T : 5'-ATT GGA TCC AGG CCG CTC TGG ACA AAA TAT GAA T r c (T)24-3' ALl : 5'-ATT GGA TCC AGG CCG CTC TGG ACA AAA TAT GAA TTC-Y Note: The ALl primer contains an EcoRI site at the 3' end and will be subsequently used for subcloning into ZZAP II phage vector. 9. Taq polymerase reaction mix 1:

Double distilled H20 5x PCR buffer 1 M TMAC ALIT primer (20 pmol/ttl) 10 mM Each dNTPs mix Taq polymerase (5U//zl) 3C. Dulac and R. Axel, Cell 83, 195 (1995).

36.65/zl 10/zl 0.4/zl 1.45/zl 1/zl 0.5/zl

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285

10. Taq polymerase reaction mix 2: Double distilled H20 5x PCR buffer 1 M TMAC AL1T primer (1 pmol//zl) AL1T primer (20 pmol//zl) 10 mM Each dNTPs mix Taq polymerase (5U//zl)

34.6/zl 10/zl 0.4 #1 1/zl 2.5/zl 1/zl 0.5 #1

11. Taq polymerase reaction mix 3: Double distilled H20 5x PCR buffer 1 M TMAC ALl primer (20 pmol//zl) 10 mM Each dNTPs mix Taq polymerase (5U//zl)

35.6/zl 10 #1 0.4/zl 2.5/zl 1/zl 0.5/zl

Methods

Laser Capture Microdissection For tissue procurement and sectioning we refer the reader to Ref. 1, and for laser capture microdissection to other chapters in this volume. One hundred to 300 laser beam shots of approximately 30-50/zm were made with the Laser Capture Microdissection instrument to transfer selected cells to the polymer film (in one cap).

RNA Isolation RNA extraction was performed using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). A 50/zl final volume of denaturing solution without addition of 2-mercaptoethanol was used per cap and stored in tubes at - 7 0 ° until ready for RNA extraction. Just before the extraction, following the procedures of the RNeasy Mini Kit, 2-mercaptoethanol was added.

cDNA Synthesis The method outlined here was adapted from Ref. 2. Shown in square brackets are the names of the programs (detailed below) in the PTC-100 programmable thermal controller (MJ Research, Inc., Waltham, MA) used to perform the procedures in the protocol. The actual writing of the programs is only a suggestion

286

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as they may change depending on the thermocycler and not for every step is it absolutely necessary to use the thermocycler. 1. Resuspend RNA in 25 #1 RNase-free water. 2. Heat at 65 ° for 2 min [LCM65] then cool on ice. 3. Put 20/zl (100/zg) of Dynabeads in 0.5-ml centrifuge tube and place it in the magnetic stand. 4. Remove supernatant. 5. Remove from stand and resuspend in 25 #1 of 2× binding buffer. 6. Remove buffer using magnetic stand. 7. Add 25/zl of 2× binding buffer to beads. 8. Add 25/zl of RNA from step 2 to beads (total volume now 50 #1) and incubate for 15 min at 22 ° [LCM22]. 9. Remove supernatant using magnetic stand. 10. Wash beads twice with 50 #1 of washing buffer and remove supernatant. 11. Wash beads in 50/zl of 2× first-strand buffer and remove supernatant. 12. Add 19/zl of reverse transcriptase mix and heat at 37 ° for 2 min [LCMRT]. 13. Add 1/zl of reverse transcriptase and mix, continue with [LCMRT]. 14. Continue incubation at 37 ° for 15 min, then increase temperature to 42 ° for 45 min (mixing tube every 15 min) and heat at 65 ° for 10 min to inactivate the reverse transcriptase [LCMRT]. 15. Remove buffer and add 20/zl of T4 DNA polymerase reaction mix. Incubate at 16 ° for 1 hr and inactivate by heating at 74 ° for 10 min [LCMTRIM]. 16. Remove buffer and add 20/zl of RNaseH reaction mix. Incubate at 37 ° for 1 hr [LCM37H], remove buffer, then add 50/zl of 1 m M EDTA and heat at 75 ° for 5 min [LCM75IH]. Remove EDTA solution. 17. Add 20/zl of Terminal Transferase mix. Incubate at 37 ° for 15 rain, then stop by adding 2/zl of 0.5 M EDTA and remove buffer [LCMTr]. 18. Add 50/zl of Taqpolymerase reaction mix 1 (containing 29 pmol of AL1T primer). Carry out annealing and extension at 30 ° for 3 min, 40 ° for 3 min, and 72 ° for 5 min [LCM2ST]. Remove supernatant. 19. Add 50/zl of Taq polymerase reaction mix 2 (containing 50 pmol ALl primer and 1 pmol AL1T primer) and heat at 95 ° for 2 min [LCM95], then transfer supernatant to another vial and save beads for future use (suggested 70% ethanol at 4°). 20. Add oil to the new vial if necessary, incubate at 30 ° for 15 min, 40 ° for 15 rain, 72 ° for 15 min, then heat at 95 ° for 2 min and amplify 15 cycles 95 ° for 1 min, 72 ° for 5 min with final extension at 72 ° for 30 min [LCMAMP]. (These conditions may change depending on the primers and polymerase being used.) 21. For gel electrophoresis analysis, if sample from final amplification is not seen, then reamplify by adding 5/zl of the reaction in Taq polymerase reaction

[24]

GENE DISCOVERY WITH L C M

287

m i x 3 (95 ° for 1 min, 72 ° for 5 m i n 15 times, and final extension at 72 ° for 7 min) [LCMRE]. 22. P r o c e e d with subcloning into an appropriate vector or use the ds c D N A for labeling to be used as a probe.

Programs LCM65 1. T = 65 °, t = 2 min 2. T = 22 °, t = c~ LCM22 1. T = 22 °, t = 15 min LCMRT 1. T = 37 °, t = 2 min (add reverse transcriptase after this incubation) 2. T = 37 °, t = 15 min 3. T = 42 °, t = 45 min (mix every 15 min) 4. T = 70 °, t = 10 min 5. T = 1 6 ° , t = c ~ LCMTRIM 1. T = 1 6 ° , t = l h r 2. T = 7 4 ° , t = 1 0 m i n 3. T = 3 7 ° , t =~ LCM37H 1. T = 3 7 ° , t = l h r LCM75IH 1. T = 7 5 ° , t = 5 min 2. T = 3 7 ° , t = oo LCMTT 1. T = 3 7 ° , t = 1 5 m i n 2. T = 3 0 ° , t = oo LCM2ST 1. T = 3 0 ° , t = 3 m i n 2. T = 4 0 ° , t = 3 min 3. T = 7 2 ° , t = 5 m i n LCM95 1. T = 9 5 ° , t = 2 m i n 2. T = 3 0 ° , t = c~ LCMAMP 1. T = 3 0 ° , t = 1 5 m i n 2. T = 4 0 ° , t = 1 5 m i n 3. T = 7 2 ° , t = 1 5 m i n 4. T = 9 5 ° , t = 2 m i n

288

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5. T = 9 5 ° , t = l m i n 6. T = 72°,t = 5 min 7. Go to 5 14 times (or more as needed) 8. T = 7 2 ° , t = 3 0 m i n 9. T = 4 ° , t = ~ LCMRE 1. T = 95 °, t = 2 min (hot start) 2. T = 95 °, t = 1 min 3. T = 72°,t = 5 min 4. Go to 2 14 times 5. T = 72°,t = 7 m i n 6. T = 4 ° , t = oo

Phage )~ZAPH Library Construction The ds cDNAs obtained with the former protocol are flanked with the ALl primer which can be cut out of the library by restriction with EcoRI. The EcoRI restricted ds cDNAs are further purified with QIAquick PCR purification kit (Qiagen Inc.) and subcloned into )~ZAP II vector (Stratagene, La Jolla, CA) following the protocol from the company.

Differential Screening Approximately 12,000 clones from the taste bud library were transferred to duplicate 150-mm round nylon filters (~2000 clones per filter), which were separately prehybridized at 68 ° for 1 hr in 6× SSC + 1× Denhardt's (Sigma, St. Louis, MO) + 40 mg/ml poly A + (Boehringer Mannheim Corp.) and then hybridized at 68 ° overnight with either the 32p-labeled (with Random Primed DNA Labeling Kit, Boehringer Mannheim Corp.) total taste bud or control epithelial cell cDNAs (1.3-2 × 10 6 cpm/ml) in 6× SSC + 1× Denhardt's + 500 mg/ml poly A+. Washing was performed three times, for 30 min each, at 68 ° in 0.5× SSC + 0.5% SDS and then exposed for 1-3 days to Xar-5 films (Eastman Kodak Company, Rochester, NY) with intensifying screens at - 7 0 °. Plaques picked on the first screen were screened a second time in the same fashion. Phage from plaques giving a signal with the taste bud-specific cDNA probe, but not with the probe from control epithelium, were processed by plasmid in vivo excision into pBluescript S K ( - ) phagemid vector (as recommended in the ~.ZAP II phage vector kit), and the plasmids were analyzed by automatic fluorescence sequence analysis. Similarly, a random set of the clones not showing hybridization with either probe can be analyzed by sequencing and chosen for primer design for subsequent PCR analysis. Note: Poly A + is added to suppress any unwanted hybridization due to poly(dT) stretches present in the cDNAs probes.

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Concluding Remarks The use of oligo(dT) magnetic beads in the cDNA synthesis allows the storage of a cell-specific ss cDNA population that can be used later for amplification. In our hands it was possible to use the beads 2-3 times more. It is important to be aware of the possible cross-cell contamination when performing laser capture microdissection. The quality of staining of the frozen sections may be crucial for the recognition of the cell population of interest and this will influence the purity of the cDNAs to be obtained. Minute cross-cell contamination may lead to contradictory results when a gene specific for a cell population shows a PCR signal in the cDNAs from this cell population and in the cDNAs from surrounding cells at the same time when differential hybridization and RNA in situ hybridization shows expression only in the specific cell population of interest. The same caution applies when analyzing clones that do not show a signal with either probe on differential hybridization and are further analyzed by PCR.

Acknowledgments We are especially grateful to Dr. Robert F. Bonner from NIH for the scientific advice in using LCM and giving us access to the NIH LCM Core Lab.

[251 DNA Fingerprinting from Cells Captured by Laser Microdissection By YONGYUT SIRIVATANAUKSORN, VORAPAN SIRIVATANAUKSORN, and

NICHOLAS R. LEMOINE

Introduction There have been dramatic advances in our knowledge of the molecular processes involved in human diseases, but it is certain that other molecular and genetic lesions remain to be identified, and there is a pressing need to integrate such information with structural and architectural data derived from conventional morphological approaches. It is obviously an advantage to use microdissected cell samples in molecular analysis because the confounding effect of contaminating cells is eliminated. Laser-assisted microdissection is one of the most advanced techniques and has been rapidly developed to procure precisely the cells of interest from complex normal and diseased tissues. Precise microdissection of phenotypically



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Concluding Remarks The use of oligo(dT) magnetic beads in the cDNA synthesis allows the storage of a cell-specific ss cDNA population that can be used later for amplification. In our hands it was possible to use the beads 2-3 times more. It is important to be aware of the possible cross-cell contamination when performing laser capture microdissection. The quality of staining of the frozen sections may be crucial for the recognition of the cell population of interest and this will influence the purity of the cDNAs to be obtained. Minute cross-cell contamination may lead to contradictory results when a gene specific for a cell population shows a PCR signal in the cDNAs from this cell population and in the cDNAs from surrounding cells at the same time when differential hybridization and RNA in situ hybridization shows expression only in the specific cell population of interest. The same caution applies when analyzing clones that do not show a signal with either probe on differential hybridization and are further analyzed by PCR.

Acknowledgments We are especially grateful to Dr. Robert F. Bonner from NIH for the scientific advice in using LCM and giving us access to the NIH LCM Core Lab.

[251 DNA Fingerprinting from Cells Captured by Laser Microdissection By YONGYUT SIRIVATANAUKSORN, VORAPAN SIRIVATANAUKSORN, and

NICHOLAS R. LEMOINE

Introduction There have been dramatic advances in our knowledge of the molecular processes involved in human diseases, but it is certain that other molecular and genetic lesions remain to be identified, and there is a pressing need to integrate such information with structural and architectural data derived from conventional morphological approaches. It is obviously an advantage to use microdissected cell samples in molecular analysis because the confounding effect of contaminating cells is eliminated. Laser-assisted microdissection is one of the most advanced techniques and has been rapidly developed to procure precisely the cells of interest from complex normal and diseased tissues. Precise microdissection of phenotypically



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similar tissue samples revealed genetic heterogeneity.1 An increase in sensitivity of more than 50% in allelic imbalance analysis was obtained by using microdissected cell populations compared with crushed frozen tumor samples. 2 The fundamental advantage of this technique is the possibility of exploiting capture on a single-cell basis and isolating high quality DNA and mRNA for analysis of sequence and quantitation of expression. In cancer models, laser-assisted microdissection provides the capacity for isolating cells from specific stages of tumorigenesis, including normal, precancerous, malignant, and metastatic cells. This will allow us to define the genetic changes associated with functional state, malignant transformation, tumor progression, tumor heterogeneity, and clonal progression. Polymerase chain reaction (PCR)-based methods for nucleic acid detection and fingerprinting have become vital to modern molecular genetics, whether for the analysis of populations of organisms to determine population structure of an ecosystem, sampling a set of DNA sequences to infer evolutionary history, sampling genetic loci to build a map, or sampling differentially expressed genes to identify phenotypic markers. PCR can be used to generate high resolution genetic maps of human and comparative genomes. The classic approach to DNA fingerprinting utilizes variable number tandem repeat (VNTR) polymorphism in which alleles differ by a variable number of tandem repeats. Although the term VNTR could encompass a wide range of repeat lengths, it is usually reserved for moderately large arrays of a repeat unit that is typically in the 5- to 64-bp region. If the VNTR locus is a member of a repeated DNA family, the use of a VNTR probe will produce a complex polymorphic band pattern on hybridization. The hybridizing bands appear on the filter as a ladder of bands, referred to as the DNA fingerprint, which visually resembles the bar codes used by stores to identify and price merchandise. Arbitrarily Primed PCR The arbitrary primer-based DNA amplification technique has been proposed as an altemative targeting tool for genetic typing and mapping. This strategy uses randomly generated primers to initiate amplification of discrete but arbitrary portions of the genome. Arbitrarily primed PCR (AP-PCR) is one of the fingerprinting techniques described by Welsh and McClelland. 3 This technique is a modification of PCR, a method that is widely used to copy sections of DNA for identifying gene structure or matching tissue specimens. PCR uses two primers whose complementary sequences flank the desired sequence to amplify a region of DNA.

l C. A. Macintosh, M. Stower,N. Reid, and N. J. Maitland, CancerRes. 58, 23 (1998). 2 H. E. Giercksky,L. Thorstensen, H. Qvist, J. M. Nesland, and R. A. Lothe, Diagn. Mol. Pathol. 6, 318 (1997). 3j. Welsh and M. McClelland, Nucleic Acids Res. 18, 7213 (1990).

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The primers usually have specific nucleotide sequences that bind to previously identified segments of DNA. They bind to specific sites on opposing strands of the double-stranded DNA and are extended by a thermostable DNA polymerase to make millions of copies of the intervening stretch of DNA. Normally, the primers are annealed to the template DNA at relatively high stringency. High stringency during the primer-annealing step ensures that the primers do not interact with the template DNA at positions where they do not match. By contrast, AP-PCR allows the detection of polymorphisms without prior knowledge of nucleotide sequence. It is based on the selective amplification of genomic sequences that, by chance, are flanked by adequate matches to an arbitrarily chosen primer. The method utilizes short primers of arbitrary nucleotide sequence (10 to 20 bases) that are annealed in the first few cycles of PCR at low stringency. The low stringency of the early cycles ensures the generation of products by allowing priming with fortuitous matches or near-matches between primers and template. This approach results in a high number of products having the original primer sequence at both ends. After a few low-stringency cycles, the annealing temperature is raised and the reaction is allowed to continue under standard, high-stringency PCR conditions. This step will amplify a discrete number of sequences among those initially targeted and permits the unbiased analysis of the cell genome. Alternatively, an intermediate stringency primer-annealing step may be used throughout the PCR to achieve a similar outcome. AP-PCR products are resolved on polyacrylamide gels and are detected by autoradiography. If two template genomic DNA sequences are different, their AP-PCR products display different banding patterns. Such differences can be exploited in ways largely analogous to the uses of restriction fragment length polymorphisms, including genetic mapping, taxonomy, phylogenetics, and the detection of mutations. AP-PCR permits the rapid and cost-effective detection of polymorphisms and genetic markers in a variety of experiments.4 Moreover, it is dramatically easier and faster than established methods of genetic mapping. The reproducible and semiquantitative amplification of multiple sequences provides a powerful tool for studying somatic genetic alterations in tumorigenesis. Peinado and colleagues showed the ability of AP-PCR to detect both qualitatively and quantitatively and to isolate, in a single step, DNA sequences representing two of the genetic alterations that underlie the aneuploidy of colorectal cancer cell, i.e., losses of heterozygosity and chromosomal gains. 5 Moreover, they confirmed that AP-PCR could yield information on the overall chromosomal composition of the cell. The intensities of the bands derived from single-copy sequences were 4 j. G. Williams, A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey, Nucleic Acids Res. 18, 6531 (1990). 5 M. A. Peinado, S. Malkhosyan, A. Velazquez, and M. Perucho, Proc. Natl. Acad. Sci. U.S.A. 89, 10065 (1992).

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proportional to the concentration of the target sequences. The outstanding result using AP-PCR fingerprinting in the field of cancer research was the discovery of the microsatellite mutator phenotype mechanism for carcinogenesis in colonic and lung cancers. 6,7 AP-PCR is also useful for the detection and isolation of DNA sequences to levels well below the minimum levels required by other available methods, 8 and the products can be used to clone or hybridize back to digested genomic DNA. 9 Unbiased DNA fingerprinting by AP-PCR is a powerful molecular approach for the cytogenetic analysis of solid tumors. It detects both gains of chromosomal regions, reflecting the presence in these regions of cancer genes, and losses of genes with negative role in cell growth. Nevertheless, the finding that DNA sequences have undergone heterozygous deletions or gains of extra copies in tumor relative to normal tissue does not ensure that these sequences are linked to genes playing an active role in oncogenesis because of the high level of random genetic damage in the genome of solid tumors. Moreover, AP-PCR is an uncomplicated and effective method for scanning the genomes of tumor samples to show the genomic heterogeneity and the evolution of differences.I°'11 Laser Capture Microdissection The laser capture microdissection (LCM) system has been developed by Emmert-Buck et al.12 and comprises a novel membrane-based microdissection technique. The system has been subsequently commercialized and used in many laboratories. A thermoplastic ethylene vinyl acetate transfer film containing a near-infrared absorbing dye attached to a 6-mm diameter rigid, fiat cap is placed in contact with a routinely prepared, hematoxylin and eosin stained tissue section. The isolation of cells from immunohistochemical or molecule-specific, fluorescenfly labeled sections improves sample imaging and can help in obtaining specific cell populations more precisely. 13 The film over the cells of interest is precisely 6 y. Innov,M. A. Peinado, S. Malkhosyan, D. Shibata, and M. Perucho, Nature 363, 558 (1993). 7 y. Anami, T. Takeuchi, K. Mase, J. Yasuda, S. Hirohashi, M. Perucho, and M. Noguchi, Int. J. Cancer 89, 19 (2000). 8 I. B. Roninson, J. E. Chin, K. G. Choi, E Gros, D. E. Housman, A. Fojo, D. W. Shen, M. W. Gottesman, and I. Pastan, Proc. Natl. Acad. Sci. U.S.A. 83, 4538 (1986). 9 C. S. Wesley, M. Ben, M. Kreitman, N. Haga, and W. E Eanes, Nucleic Acids Res. 18, 599 (1990). 10 y. Sirivatanauksom, V. Sirivatanauksorn, S. Bhattacharya, B. R. Davidson, A. E Dhillon, A. K. Kakkar, R. C. N. Williamson, and N. R. Lemoine, Gut 45, 761 (1999). 11 y. Sirivatanauksorn, V. Sirivatananksorn, S. Bhattacharya, B. R. Davidson, A. E Dhillon, A. K. Kakkar, R. C. N. Williamson, and N. R. Lemoine, J. PathoL 189, 344 (1999). 12 M. R. Emmert-Buck, R. E Bonner, E D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 13 E Fend, M. R. Emmert-Buck, R. Chuaqui, K. Cole, J. Lee, L. A. Liotta, and M. Raffeld, Am. J. Pathol. 154, 61 (1999).

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activated by a near-infrared laser pulse and bonds strongly to the selected cells. Removal of the cap from the tissue section effectively procures the targeted cells. The identity of the transferred cells attached to the film can then be recorded by image capture. The cap is lifted off the tissue and placed directly onto a 0.5-ml microfuge tube containing 50/zl of proteinase K buffer. The tube is inverted and incubated overnight at 37 °. After the incubation period, the tube is centrifuged at 10,000g for 5 min and the cap is removed. Then the buffer is inactivated at 95 ° for 10 min and the sample is ready to use as a template for PCR. To examine the quality of the DNA samples from the microdissection technique, 4/zl of DNA solution is used for amplification with GAPDH primers in a total volume of 50/zl solution containing 50 mM KC1, 10 mM Tris-HC1 pH 8.3, 1.5 mM MgC12, 100 pM primers, and 5 units Taq polymerase. Templates are denatured for 5 min at 95 ° and are subject to 35 cycles at 94 °, 1 min; 55 °, 1 min; and 72 °, 2 min. The PCR products are run on 1.5% agarose gel staining with ethidium bromide. Then the DNA fingerprint from the cells of interest is amplified by the AP-PCR technique. Arbitrarily P r i m e d P C R Materials 1. Stocks of all four dNTPs (5 mM) 2. Stock of arbitrary primer (100 ~M) 3. Radioisotope ([ot-32p] or [y_33p]) dATP (>2500 Ci/mmol) 4. Taq polymerase (5 U//zl) 5. Formamide dye solution: 96% formamide, 0.1% bromphenol blue, 0.1% xylene cyanol, 10 mM EDTA 6. 10x Tris-borate-EDTA (TBE) buffer: 90 mM Tris-borate, 20 mM Na2EDTA, pH 8.3 7. Acrylamide stock solution [40% acrylamide : bisacrylamide (29 : 1)] 8. Thermocycler (e.g., Peltier Thermal Cycler, model PTC-100) 9. Sequencing gel electrophoresis apparatus (e.g., 40 cm long, 30 cm wide, 0.4 mm thick) 10. Gel dryer 11. X-ray film and exposure cassette Me~o~ A. Polynucleotide Kinase Reaction. This procedure is useful for radioactive labeling of the 5' end of oligonucleotide primers. The kinase enzyme requires that the 5' end of the oligonucleotide has been previously dephosphorylated with

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alkaline phosphatase. The forward kinase reaction catalyses the exchange of the terminal y-phosphate, which is labeled with 33p from the ATP to the terminal phosphate on the oligonucleotide. 1. Mix 100 pM of oligonucleotide with 50 mM Tris-C1 pH 7.5, 10 mM MgC12, 5 mM DTr, 0.5 mM spermidine, 50 Ci of [y-33p]dATP, and 10 U of T4 polynucleotide kinase enzyme. 2. Incubate the reaction for 1 hr at 37 °.

B. Arbitrarily Primed PCR 1. Mix template DNA (100 ng) with reaction mixture for a 25-/zl final reaction containing [y-33p]ATP-labeled and kinased arbitrary primer, 0.2 mM each dNTP (Bioline), 10 mM Tris-Cl pH 9.2, 3.5 mM MgCI2, 75 mM KC1, and 0.5 U of Taq DNA polymerase. 2. Perform thermocycling using 5 low-stringency steps, followed by 35 highstringency steps as follows: 5 low-stringency cycles (94 ° for 1 min; 50 ° for 5 rain; 72 ° for 5 min) then 35 high-stringency cycles (94 ° for 1 min; 60 ° for 1 rain; 72 ° for 2 rain) and A final chase cycle of 72 ° for 5 min to allow complete elongation of all products (After 5 low-stringency cycles, exact copies of the primer sequence flank a handful of anonymous sequences. Thus, the annealing temperature can be raised after a few cycles and the reaction allowed to continue under standard, high-stringency PCR conditions. The two-step low-high stringency protocol was designed to avoid internal priming within a larger amplifying product.) 3. Mix amplification product with 5 #1 of formamide dye solution, denature at 95 ° for 3 min, and load 5/zl of mixture onto an 8% acrylamide gel matrix prepared in I x TBE buffer. 4. Perform electrophoresis using a Sequencing Gel Electrophoresis Apparatus at 12 W, constant voltage overnight. 5. After electrophoresis, transfer the gel to Whatman 3MM paper, and dry under vacuum at 80 ° for 2 hr. 6. Autoradiograph the dried gel using an X-ray film at room temperature overnight or for 2-3 days as required.

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[261 Single Cell PCR in Laser Capture Microscopy By SINUHEHAHN, XIAO YAN ZHONG, and WOLFGANGHOLZGREVE Introduction The ability to analyze single cells by PCR has opened up many new avenues for diagnosis and research. One of the most prominent diagnostic applications of this technology currently is preimplantation genetic diagnosis (PGD)J '2 In this examination one or two embryonic cells are biopsied from human preimplantation embryos. An advantage of this procedure is that it is fairly simple to obtain single embryonic cells free from any potential paternal (spermatozoa) or maternal (cumulus cells) contaminants. Another potential diagnostic application of single cell PCR is the examination of single fetal erythroblasts retrieved from maternal circulation, where, by a combination of enrichment and micromanipulation, it is possible to isolate the desired single fetal cell from a relatively large host of coenriched maternal cells. 3 This is in stark contrast to the isolation of single cells from tissue sections, where conventional micromanipulation techniques, of the type applied for PGD or isolation of fetal cells, are wholly unsatisfactory. To circumvent these limitations several laser-based tools have been devised for the reliable and effective retrieval of single cells from complex preparations. Laser Capture Microdissection The laser capture microdissection (LCM) system was developed by Liotta and colleagues4 at NIH (the National Institutes of Health, Bethesda, MD) and is marketed by a spin-off company, Arcturus Engineering (www.arctur.com), under the name PixCell. The LCM system employs a 980 nm IR diode laser mounted above the microscope stage, which is pulsed through a special optical quality cap the size of a standard PCR reaction vessel. The base of this cap is lined with a 5-/zm synthetic transfer membrane which is brought to rest on the microscope slide with the tissue preparation. The application of a pulse of laser energy, which is focused on the desired target cell, melts the thermoplastic membrane, thereby covalenfly bonding the target cell to the PCR reaction vessel cap. This process can then be repeated to permit the collection of numerous singly isolated cells, or the cap can 1 A. H. Handyside, Prenat. Diagn. 18, 1345 (1998). 2 S. Hahn, X. Y. Zhong, C. Troeger, R. Burgemeister, K. Gloning, and W. Holzgreve, Cell. Mol. Life Sci. 57, 96 (2000). 3 W. Holzgreve and S. Hahn, Clin. Perinatol. 28, 353, ix (2001). 4 M. R. Emmert-Buck, R. E Bonner, P. D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996).

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be placed on a standard-sized PCR reaction vessel for further molecular biological analysis. Since its inception, 4 this tool has gained rapid acceptance in many research and diagnostic circles, 5,6 most prominently in those centers interested in tumor heterogeneity, 7 cellular differentiation, 8 and preimplantation genetic diagnosis, 9 as well as cell-specific drug interactions) ° These studies have used PCR, RTPCR, real-time PCR, and gene expression profiling by microarray analysis. It is worth noting that although LCM in theory is capable of isolating single cells, almost all of these numerous studies have been performed using pools of cells which had been singly captured. This partly stems from the inability to focus the laser sufficiently finely to accommodate the size and shape of single cells, tl Furthermore, when a membrane-lined PCR cap is placed over the entire specimen, it is very likely that cells or cell debris can easily become attached to this membrane, thereby contaminating the captured single cell preparation. Although this can be overcome to some extent by blotting the membrane-lined cap on a piece of sterile adhesive tape to remove noncovalently bonded cells, this approach is less than satisfactory for work requiring high degrees of purity. The other reason may be that the reliability of single cells from PCR is still technically challenging. 2 In order to facilitate the better capture of single cells, two approaches have been taken. In the first, a high-tech approach developed by NIH, a computer-controlled arm carefully positions a 40-/zm wide strip of the thermoplastic membrane with a very light contact force on the specimen. 11 A modified laser is then used to epi-irradiate individual cells with highly focused rapid laser pulses. This system uses computer-assisted rotation of a membrane-coated cylinder, whereby it is possible to capture multiple single cells individually. By the use of this approach, it is reasoned that highly selective transfer of single cells is possible. Unfortunately, this system is so complex that it is not yet commercially available. Arcturus, the company marketing the L C M system, has taken a rather low-key approach, developing a special cap (termed CapSure HS noncontact caps) used for the capture procedure whereby only a very small membrane area is in contact with the specimen. The usefulness of this in practice still needs to be verified in large-scale studies. 5 M. A. Rubin, J. Pathol. 195, 80 (2001). 6 j. C. Mills, K. A. Roth, R. L. Cagan, and J. I. Gordon, Nat. Cell BioL 3, E175 (2001). 7 F. Fend and M. Raffeld, J. Clin. Pathol. 53, 666 (2000). 8 H. Ohyama, X. Zhang, Y. Kohno, I. Alevizos, M. Posner, D. T. Wong, and R. Todd, BioTechniques 29, 530 (2000). 9 A. Clement-Sengewald,T. Buchholz, and K. Schiitze, Pathobiology 68, 232 (2000). 10T. Betsuyaku, G. L. Griffin,M. A. Watson, and R. M. Senior, Am. J. Respir. Cell. Mol. BioL 25, 278 (2001). 11 C. A . Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, and R. E Bonner, BioTechniques 26, 328 (1999).

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Laser Pressure Catapulting Biological application of laser pressure catapulting (LPC) was pioneered by Schtitze and colleagues 12'13 and developed at the same time as the LCM system (www.palm.microlaser.com). This system also uses an inverse microscope, but uses an epifluorescence laser system instead of an overhead laser, In LPC, a 337 nm high quality pulsed nitrogen laser is focused through the microscope optics onto the specimen and with the use of high numerical long-distance objectives a spot size of 2 /zm can be attained. The LPC system differs from the LCM system in that the cell preparation is transferred onto a glass slide covered with a thin polyethylene membrane. Once the desired target cells have been localized, they are first excised by the high energy laser beam and then in a second step are catapulted by a focused pulse of laser energy directly into the cap of a PCR reaction vessel suspended above the microscope slide. The use of the synthetic membrane permits retention of the target cells during the microdissection and catapulting steps. Advantages of this system are the ability to finely focus the laser beam and the fact that the high energy laser can be used to ablate cells adjacent to the target cell. It therefore appears better suited to the isolation of single cells than the current LCM version. Comparative Analysis As our interest lies in the reliable retrieval of single fetal cells from maternal circulation, 3 we have performed a comparative analysis in which we investigated the efficacy of LCM and LPC systems and classical manual micromanipulation. In this study, cells were transferred onto glass slides by cytocentrifugation and histochemically stained. Single lymphocytes were isolated by the three different microdissection methods and examined by a nested PCR procedure for the ubiquitous/%globin gene which had been optimized for the analysis of single cells.14,15 This control locus had previously been shown to be very effective in monitoring the efficacy of the PCR reaction and in ascertaining whether a single cell had indeed been transferred to the reaction vessel. 16 In our study a total of 359 single cells were analyzed: 61 by LCM, 99 by LPC, and 199 by capillary-based micromanipulation. The single cell PCR analysis indicated that the fl-globin gene could be readily detected in 52% of the cells isolated by LCM, 56% of the cells isolated 12 K. Schiitze, A. Clement-Sengewald, and A. Ashkin, Fertil. Steril. 61, 783 (1994). 13 K. Schtitze, I. Becker, K. E Becker, S. Thalhammer, R. Stark, W. M. Heckl, M. Bohm, and H. Posl, Genet. Anal. 14, 1 (1997). 14 C. Troeger, X. Y. Zhong, R. Burgemeister, S. Minderer, S. Tercanli, W. Holzgreve, and S. Hahn, Mol. Hum. Reprod. 5, 1162 (1999). 15 X. Y. Zhong, W. Holzgreve, J. C. Li, K. Aydinli, and S. Hahn, Prenat. Diagn. 20, 838 (2000). 16 S. Hahn, X. Y. Zhong, M. R. Burk, C. Troeger, and W. Holzgreve, Ann. N.Y Acad. Sci. 906, 148 (2000).

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by LPC, and 43% of those retrieved by manual micromanipulation. It, therefore, appears that the isolation of single cells by either of the laser-based systems is more effective than by traditional methods. A major factor to consider here is the reliable transfer of the isolated single cell into the PCR reaction vessel, which appears to be better safeguarded by the laser-mediated tools than by use of a finely drawn capillary, from which it may be more easily lost. Current Limitations of Individual Methods Although it may appear that the LPC system is better suited than current LCM tools for the retrieval of single cells, both systems have their individual advantages and disadvantages. As mentioned above, LCM of single cells is currently hampered by the use of a rather large membrane surface covering a significant portion of the target cell preparation. Since this membrane is in direct contact with the specimen, it is difficult to be certain that no extraneous genetic material has been introduced into the final single cell preparation. This large membrane surface also hinders the effective analysis of single cells, as it is very difficult to extract the DNA from the single cell attached to this membrane in a sufficiently small volume of extraction buffer which still permits an effective PCR analysis. Indeed, in our experience, it was not possible to reliably isolate the single cell DNA by moistening the membrane while attached to the cap; rather, the membrane had to be removed and immersed in a separate PCR reaction vessel. This additional step was not only tedious, but a significant potential source of contamination. It is to be hoped that the use of new noncontact caps will alleviate this problem, until the more complex cylinder rotation system becomes commercially viable. On the other hand, we have determined that the membrane used for the LPC system, although important for the maintenance of cell integrity, does compromise the use of this system. In this regard, we have experienced that while the membrane coated slides are very suitable for use with microtome-cut tissue sections, they are less than ideal with cytocentrifuged cell preparations. This is due to the lack of adequate adhesion of cell suspensions to this membrane, which results in significant cell loss during staining procedures. Although this is of little concern when dealing with an abundant cell type, it is a definite problem when investigating rare events, such as fetal cells enriched from the maternal circulation. Staining Procedures Including Fluorescence-Based Methods A further problem we have encountered is that LCM is adversely affected by the majority of histological staining procedures, such as May-Gruenwald Giemsa, which are commonly used for the initial distinction of various cells types.14'15 This is apparently because the preparation is not sufficiently dehydrated to permit effective transfer of the laser energy through the target cell to achieve its

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thermobonding to the carder membrane. The use of extra dehydration steps has the effect of leaching the desired dyes from the preparation, thereby rendering identification of the desired target cell impossible. Of the diverse histological and immunohistochemical staining procedures we have used for the putative identification of fetal erythroblasts, we determined that 3,3'-diaminobenzidine (DAB) based stains were optimal. It is unclear how this physical need for extremely dry preparations will affect the use of fluorescent immunohistochemical protocols. We have, however, determined that the use of FISH (fluorescence in situ hybridization) protocols does prevent the effective retrieval of target cells by LCM. Consequently, when considering cell recycling approaches, other strategies have to be chosen in which individual cells are first analyzed by FISH to determine chromosomal ploidy and subsequently by PCR to examine for the presence of a specific inherited single gene disorder. 2,17 In the same manner, we have noted that the use of membrane-coated slides for LPC severely restricts the use of fluorescence-based approaches because of the very high level of autofluorescence of this membrane. Furthermore, as the laser of the LPC system uses the same pathway as that of the epifluorescence system, the fluorescence intensity in such modified microscopes is dramatically reduced, particularly for certain wavelengths, such as those needed to excite the DAPI channel. We have not been able to optimize this system to the extent that it can be used for cell recycling approaches. It is clear that it will also need to be optimized carefully when using fluorescent immunohistochemistry for the identification of potential target cells. RT-PCR and Microarray Analysis Even though several studies using laser microdissection have focused on the detection of genetic mutations, such as the presence of malignant cells in a given tissue, a considerable body of work has been performed examining gene expression using this technology.8,18-21 One aspect worth noting with regard to these studies is that these studies are almost exclusively performed on pools of cells. Several of these studies have employed cutting-edge technology, such as real-time PCR, 22 or

17 A. Sekizawa, O. Samura, D. K. Zhen, V. Falco, and D. W. Bianchi, Am. J. Obstet. Gynecol. 181, 1237 (1999). 18 K. Schutze and G. Lahr, Nat. Biotechnol. 16, 737 (1998). 19 I. Alevizos, M. Mahadevappa, X. Zhang, H. Ohyama, Y. Kohno, M. Posner, G. T. Gallagher, M. Varvares, D. Cohen, D. Kim, R. Kent, R. B. Donoff, R. Todd, C. M. Yung, J. A. Warrington, and D. T. Wong, Oncogene 20, 6196 (2001). 2o M. Neira, V. Danilova, G. Hellekant, and E. A. Azen, Mature. Genome 12, 60 (2001). 21 K. E. Dolter and J. C. Braman, BioTechniques 30, 1358 (2001). 22 L. Fink, W. Seeger, L. Ermert, J. Hanze, U. Stahl, E Grimminger, W. Kummer, and R. M. Bohle, Nat. Med. 4, 1329 (1998).

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high density gene microarrays. 5'6'8' 19 In this regard, Fink and colleagues examined TNF-c~ expression by real-time PCR in small numbers of individually isolated alveolar macrophages obtained by LPC from bronchiolar lavage specimens. 22 LCM technology has also been used by Betsuyaku et al. to explore the action of drugs on distinct cell populations, such as bleomycin on bronchiolar epithelium by real-time PCR. 1° Furthermore, the expression profiles of LCM dissected tumor and normal epithelial cells have been examined by microarray analysis. 5'6 In one such study approximately 600 cancer-associated genes were identified. 19 Because of the number of cells required for these analyses, between 100 and 1000, considerable progress still has to be made before this technology becomes available for single cell analysis. One way of overcoming this deficit may be to use whole genome gene amplification strategies similar to those used by numerous research groups for the PCR analysis of several genetic loci from single cells. 2,23'24 Additionally, the mRNA can be linearly amplified using T7 RNA polymerase. 8 This latter approach has been shown to be suitable for generating biotinylated cRNA species which can be readily detected using high density oligonucleotide arrays. 8 Another major problem when dealing with mRNA expression studies is the preparation of the specimen. Studies have indicated that archival tissue can be readily used for DNA analyses, whereas those used for mRNA expression need to be specially prepared to prevent mRNA degradation. 25-27 In this manner cryopreserved tissues yielded a greater abundance of mRNA species than paraffin embedded sections as measured by real-time PCR for standard housekeeping genes. A further major concern is that exposing the specimen to aqueous solutions can lead to the destruction of almost 99% of the RNA. To overcome this, special rapid immunofluorescence methods have been developed in which the specimen is labeled and fixed in a 1 min procedure. 26,28-3° Results from other studies have indicated that brief counterstaining of cryosections with hematoxylin may leave cells sufficiently intact to permit an examination of several distinct mRNA species. 27

23V. G. Cheungand S. E Nelson, Proc. Natl. Acad. Sci. U.S.A. 93, 14676 (1996). 24C. P. Beltinger, E Klimek, and K. M. Debatin, Mol. Pathol. 50, 272 (1997). 25S. M. Goldsworthy,E S. Stockton,C. S. Trempus,J. E Foley, and R. R. Maronpot,Mol. Carcinog. 25, 86 (1999). 26E Fend, M. Kremer, and L. Quintanilla-Martinez, Pathobiology 68, 209 (2000). 27N. Tanji, M. D. Ross, A. Cara, G. S. Markowitz,E E. Klotman, and V. D. D'Agati, Exp. Nephrol. 9, 229 (2001). 28E Fend, M. R. Emmert-Buck, R. Chuaqui, K. Cole, J. Lee, L. A. Liotta, and M. Raffeld, Am. J. Pathol. 154, 61 (1999). 29L. Fink, Z. Kinfe, M. M. Stein, L. Ermert, J. Hanze, W. Kummer,W. Seeger, and R. M. Bohle, Lab. Invest. $0, 327 (2000). 30H. Murakami, L. Liotta, and R. A. Star, Kidney Int. 58, 1346 (2000).

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The ability to obtain small numbers of a particular cell type has facilitated the generation of well-defined cDNA libraries which permit the identification of new tissue-specific genes. 2° In this manner a taste bud specific transcript (rmSTG) was detected by differential screening from a small number of laser manipulated R h e s u s monkey taste bud cells. 2° It is to be expected that these techniques will be optimized to such an extent that it will become possible to examine gene expression differences in various cell types in a tissue section, thereby permitting a more detailed analysis of cell differentiation and the process of cancer formation. Conclusions In the few years since the development of laser-mediated microdissection technologies tremendous progress has been made in the analysis of cells captured by these means. It is clear that the analysis of single cells will always be hampered by the limiting amount of target template, be this DNA or mRNA, despite the use of strategies to amplify the template in an unbiased manner. 2'31,32 This aspect may also become more apparent when examining different cell types, such as erythroblasts, which have higher allele dropout rates than other hemopoietic cells.31 Other issues which currently need to be addressed are reliable contamination-free excision of single cells from complex specimens using LCM and methods to obviate the need for membrane-coated slides in LPC, from which rare cells are prone to be lost during the preparation of the specimen. Furthermore, effective methods will have to be developed which enable the efficient manipulation of cells labeled by immunofluorescent means, especially those which have been previously analyzed by FISH for their chromosomal complement. There is, however, little doubt that the analysis of single or few cells obtained by laser mediated microdissection is going to gain in clinical importance in the near future and that this technology will also open new, exciting avenues for research, especially for those interested in cellular differentiation, be this normal or pathological.

31A. M. Garvin,W. Holzgreve,and S. Hahn,Nucleic Acids Res. 26, 3468 (1998). 32S. Hahn, A. M. Garvin, E. Di Naro, and W. Holzgreve,Genet. Test. 2, 351 (1998).

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[27] Assessment of Genetic Heterogeneity in Tumors Using Laser Capture Microdissection By DAVE S. B. HOON, AKIHIDEFUJ1MOTO, SHERRY SHU, and BRET TABACK Introduction Assessment of genetic changes that contribute to the tumor heterogeneity within a tumor lesion is an important and technically complex problem. We have employed laser capture microdissection (LCM) to assess archived small metastatic melanoma lesions for tumor genetic changes. The focus of the study is to examine inter- and intratumor genetic heterogeneity within melanoma lesions. DNA microsatellite markers with loss of heterozygosity (LOH) are assessed in melanoma lesion sections microdissected with LCM. Both inter- and intratumor genetic heterogeneity are observed in these tumor lesions. There are now ample studies demonstrating that human solid tumors are heterogeneous in specific genetic markers or expression of individual genes. Tumor heterogeneity is the inherent problem that has significantly hampered cancer diagnosis and treatment. Tumor heterogeneity occurs at the "macro" level involving clonal cell types within a tumor lesion and at the "micro" level involving genetic differences and gene expression levels within cells of a tumor lesion. The heterogeneity of tumors at the molecular level has only recently been appreciated. This has come about through the development of improved and more sensitive molecular detection assays, and most importantly through new approaches to microdissection of a given tumor lesion and isolation of a specific group of cells. The latter have significantly evolved through the development of LCM. The LCM technique has allowed investigation of tumor lesions at the micro level, which has not previously been available to researchers. The approach has allowed more accurate, focused, and comparative analysis of defined regions or cells within a tumor lesion. The rapid evolution of molecular biology of tumors and LCM analysis has provided a very powerful analytic approach to assess tumor heterogeneity and understand tumor progression better at the molecular level. In our laboratory a major ongoing study is the assessment of genetic markers and their relevance in tumor progression such as in melanoma. The focus of our studies has been on specific multiple DNA microsatellite markers in which there is LOH. Studies from our laboratory and others have demonstrated that there are frequent LOH of specific microsatellite markers covering several major chromosome arms in melanoma.l-4 The detection of LOH at specific chromosome sites 1 T. N a k a y a m a , B. Taback, R. Turner, D. L. Morton, a n d D. S. B. H o o n , Am. J. Pathol. 158, 1371 (2001).

METHODSIN ENZYMOLOGY,VOL. 356

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in various cancers has been instrumental in the identification of tumor suppressor genes or tumor-related genes. The detection of specific LOH markers in tumor lesions has also been shown to be a very valuable prognostic marker for disease outcome. 2'5'6 Traditional methods have been to dissect out tumor lesions from paraffin-embedded sections under a micrcoscope using a scalpel or needle. This approach was basically the only practical method available until the development of LCM. The limitations of previous approaches of tumor dissection were contamination of normal cells, the size of tumor cell clumps, and the accuracy of particular cell types being dissected. It is now obvious that previous approaches may have underestimated LOH of multiple markers in a tumor lesion, and, also, the assessment of the particular site of the lesion may not be representative of the lesion as a whole. In assessment of tumor lesions for genetic markers one of the major limitations has been the size of the tumor lesion to be dissected. The majority of the studies of LOH have been on large tumor lesions that are easily dissected out, usually hematoxlyin and eosin positive-stained lesions greater than 5 mm, with a scalpel/needle with light microscopy. In recent years primary tumor lesions and metastasis have been detected at earlier stages; thus the lesions are being detected when they are smaller. Recently, there has been significant growing importance in the detection of "micrometastasis" in tumor-draining lymph nodes. The genetic changes in these early stages of metastatic tumor establishment are important in assessment. Micrometastasis is a loosely defined term that can refer to anything from a few occult tumor cells to a clump (10-100 cells) of tumor cells. The advent of LCM has allowed us to improve our dissection of these micrometastases, isolate a sufficient amount of nucleic acids, and perform molecular analyses. Obviously, the limitations are specimen preservation and the amount of nucleic acids recovered. Nevertheless, LCM has opened up a new area of the accurate assessment of the early stages of tumor metastasis. In this study we describe the utilization of LCM in the assessment of LOH markers in two types of metastatic melanoma diseases, in-transit melanomas and lymph node metastasis.

2 y. Fujiwara, D. J. Chi, H. J. Wang, E Kelemen, D. L. Morton, R. Tumer, and D. S. B. Hoon, Cancer Res. 59, 1561 (1999). 3 T. Nakayama,B. Taback,D.-H. Nguyen, D. L. Morton, and D. S. B. Hoon,Ann N. Y. Acad. Sci. 9tl6, 87 (2000). 4 R. Morita, A. Fujimoto, N. Hatta, K. Takehara, and M. Takata, J. Invest. Dermatol. 111, 919 (1998); E. Healy, C. Belgain, M. Takata, A. Vahlquist, I. Rehman, H. Rigby, and J. Rees, Cancer Res. 56, 589 (1996). 5 B. Taback, A. E. Giuliano, N. M. Hansen, and D. S. B. Hoon, Ann. N.Y. Acad. Sci. 945, 22 (2001). 6 B. Taback, Y. Fujiwara, H.-J. Wang, L. J. Foshag, D. L. Morton, and D. S. B. Hoon, CancerRes. 61, 5723 (2001).

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Materials and Methods

Specimens Primary and metastatic melanoma paraffin-embedded tissue blocks were obtained from Saint John's Health Center pathology department. All studies were under patients' written consent through an approved Human Subject's Committee IRB protocol. Control DNA for each melanoma patient was obtained from either their peripheral blood lymphocytes (PBL) or from spotted blood on FTA cards (GIBCO, NY) using the QIAamp blood kit.

LCM and DNA Extraction Paraffin-embedded tumor tissue from melanoma patients was assessed for intratumor heterogeneity using LCM. Two or three random and discrete regions from each melanoma lesion were selected for LCM and DNA extraction. Amplitude, pulse duration, and number of hits were adjusted to capture approximately 4 x 106/zm 3 of tissue. DNA was isolated with 55 /zl proteinase K (0.18 mg/ml proteinase K, 45 mM Tris-HCL pH 8.0, 0.9 mM EDTA, and 0.45% Tween 20) at 42 ° overnight, followed by heat-denature of proteinase K at 95 ° for 10 min.

Microsatellite-PCR LOH Analysis Eight markers covering six different chromosomes were selected for PCR amplification. These markers were selected because they showed a high incidence of LOH in either primary melanoma tumors or advanced metastases. Studies were conducted with FAM-labeled microsatellite markers for vertical gel electrophoresis or Beckman dye fluorolabeled dye microsatellite markers for capillary array electrophoresis (CAE). The following FAM-labeled microsatellite markers were used in this study: D1S228 at lp36; D1S314 at lp36.3; D3S1293 at 3pter3p24.2; D6S264 at 6q25.2-q27; D9S157 at 9p23-p22; D9S04 at 9p21; D10S212 at 10q26; and D11S2000 at 1 lq22-q23. The following microsatellite markers were used for CAE: D1S228; DS1293; D9S157; and D11S200. These markers were labeled with WellRed phosphoramidite-linked dye or active ester-labeled dye. PCR primer sets for specific allele loci were obtained from Research Genetics, Inc. (Huntsville, AL). Genomic DNA (~50 ng) extracted by LCM and matching PBL was amplified using PCR in a 10 #1 reaction volume, containing 15 mM Tris-HCl (pH 8.0), 50 mM KC1, 1.5 mM MgC12, 0.8 mM deoxynucleotide triphosphates, 0.25 # M forward primer, 0.25/zM reverse primer, and 0.5 U of Amplitaq Gold DNA polymerase (PerkinElmer, Norwalk, CT). PCR cycles consisted of 30 sec at 94 °, 30 sec at 50-56 ° depending on the primer sets, and 30 sec at 72 ° for a total of 40 cycles. This was followed by a 5 min final extension at 72 °. One/zl of PCR product was mixed with 40/xl of loading buffer and 0.5/zl of CEQ DNA size standards (Beckman Coulter, Inc., Fullerton, CA) in a 96-well microtiter plate.

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Sample was then electrophoresed on the CEQ 2000XL DNA Analysis System (Beckman Coulter, Inc.). The CAE system contains 8 capillaries, each with a 33 cm length (Beckman Coulter, Inc.). Samples were denatured at 90 ° for 120 sec, introduced into the capillaries by electrokinetic injection for 30 sec at 2.0 kV, and electrophoresed at 6.0 kV for 35 min. The total cycle for each row of eight samples, which included denaturation, injection, separation, data analysis, and capillary replenishment, was approximately 45 rain. Peak intensity and relative size were generated automatically by CEQ 2000 Fragment Analysis System Software (Beckman Coulter, Inc.). To estimate the degree of LOH, normalized ratios are calculated as (T1/T2)/(N1/N2) where T1 and N1 are the peak heights of the lighter alleles and T2 and N2 are the peak heights of the heavier alleles of tumor DNA (T) and PBL DNA (N). The tumor was scored as exhibiting LOH when the ratio was greater than 2.0 or lower than 0.5. For vertical gel electrophoresis the gel was scanned by a fluorescent/optical GenomyxSC scanner (Beckman Coulter, Inc.). Densitometry was performed on the gel images and analyzed using ClaritySC 3.0 software (Media Cybernetics, Silver Spring, MD). The tumor was scored as exhibiting LOH if there was a >50% reduction in signal intensity of one allele when compared to the respective allele in the corresponding normal DNA (lymphocytes) for both CAE and vertical gel electrophoresis analysis. Results and Discussion In-transit melanoma is a locoregional metastasis that often develops into an aggressive disease and eventual systemic metastasis. The disease is often characterized by rapid recurrence of local regional metastases after the primary or metastastic lesions have been removed. This melanoma is often cutaneous and recurrence is in the form of multiple nodules usually < 1 cm. This form of melanoma is very difficult to treat and remains an enigma to clinicians. The disease is also very interesting in terms of its biological behavior. There have been no major studies assessing the genetic analysis of in-transit melanomas. However, there are questions as to whether the metastases are clonal in origin. We took on the approach of assessing clonality of these in-transit metastatic and respective primary lesions by assessing commonly found microsatellites with LOH in melanomas. The use of LCM provided a unique opportunity to assess intratumor and intertumor heterogeneity. In 19 of the 25 patients (informed patients) LOH was detected for at least one of the eight microsatellite markers. The frequency of individual markers was as follows in order of frequency: D9S157 (56%); D9S304 (47%); D11S2000 (39%); D10S212 (32%); D1S214 (32%); D6S264 (22%); D1S228 (11%); and D3S1293 (10%). In examining 79 lesions from 25 patients there were only six lesions showing different LOH profiles compared to the other respective lesions of the patient. In these six lesions no significant marker stood out. We examined intratumor

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Tv missense Tv missense Tv missense Tv missense Tv missense Ts silent Ts missense Ts nonsense Tv missense Tv nonsense Ts silent Ts silent Ts missense Ts missense Ts missense Tv silent Tv silent Ts silent Ts missense Ts silent Ts missense Tv silent Ts silent Ts missense Tv nonsense Tv silent Tv silent

PIN, prostatic intraepithelial neoplasia; PCA, prostatic carcinoma; Ts, transition; Tv, transversion; T, transition zone; N-T; nontransition zone. b Mutation at CpG sites. From H. Takayama, M. Shin, N. Nonomura, A. Okuyama, and K. Aozasa, Jpn. J. CancerRes. 91,941 (2000). a

1

2

3

4

FIG. 3. PCR-single-strand conformation polymorphism (SSCP) analysis of p53 mutation. Nonradioactive SSCP analysis of exon 7. Aberrant migration patterns (arrow) were seen in lane 1 (case 3, PCA), lane 2 (case 6, PCA), and lane 3 (case 19, PIN). Wild-type SSCP bands are shown in lane 4 (case 19, benign prostatic hypertrophy).

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317

GENE MUTATIONS IN PROLIFERATIVE PROSTATIC DISEASES TABLE III SUMMARYOFp53 MUTATIONSIN PCA AND PIN Mutation frequency (%) Stage

PCA PIN

Zone

Distance from PCA

Case

Lesion

T2 a

T3 a

Transitiona

Non-transitiona

2mm -2/65 a (3.0)

ap < 0.05. From H. Takayama, M. Shin, N. Nonomura, A. Okuyama, and K. Aozasa, Jpn. J. Cancer Res. 91, 941 (2000).

Frequency of p53 mutation in PIN in the nontransition zone (14.7%) was higher than that in the transition zone (5.6%), although the difference was not significant. The frequency rate of p53 mutation in HGPIN close to PCA (2 mm of distance from PCA (3%) (p < 0.05). Information on the molecular genetic characteristics of PIN was quite limited until the development of the microdissection technique. Previous studies showed that allelic loss of chromosomes 8p, 10q, and 16q was frequent both in the HGPIN and in invasive PCA, suggesting the involvement of tumor suppressor genes or oncogenes located on these loci. 7'8'26,27 In our studies, the frequency rate of p53 gene mutations in the PCA lesions, mainly from stages T2 and T3 of the disease (20%), was close to that in the cases reported with advanced PCA. 28-3° Frequency of the p53 gene mutations in the HGPIN lesions was 12%. There is a difference in mutation frequency of PCA lesions in the different stages of the disease: one of 22 lesions (4.5%) in T2 and 10 of 33 lesions (30.3%) in T3 stage showed the mutations. This suggests that p53 gene mutations may be

26 I. C. Gray, S. M. A. Phillips, S. L. Lee, J. E Neoptolemos, J. Weissenbach, and N. Spurr, Cancer Res. 55, 4800 (1995). 27 S. E. Strup, R. O. Pozzatti, C. D. Florence, M. R. Emmert-Buck, E H. Duray, L. A. Liotta, D. G. Bostwick, W. M. Linehan, and C. D. Vocke, J. Urol. 162, 590 (1999). 28 R. Bookstein, D. MacGrogan, S. G. Hilsenbeck, E Sharkey, and D. C. Allred, CancerRes. 53~ 3369 (1993). 29 M. Watanabe, T. Ushijima, H. Kakiuchi, T. Shiraishi, R. Yatani, J. Shimazaki, T. Kotake, T. Sugimura, and M. Nagao, Jpn. J. CancerRes. 85, 904 (1994). 30 D. Mirchandani, Z. Zheng, G. J. Miller, A. K. Ghosh, D. K. Shibata, R. J. Cote, and P. Roy-Burman, Am. J. Pathol. 147, 92 (1995).

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[9,8]

involved in the progression of PCA. Berner et al. 31 reported that C-to-G transversion at codon 273 was frequently found in PCA. They suggested that this may be a mutational hot spot in the progression of PCA. Although exon 5 was the commonest site for mutations in our series, there were no mutational hot spots. Mutations at the CpG site were found in one case (case 21). G-to-A transition and C-to-G transversion substitution was most common in our series. In 8 of 12 cases with P C A and/or PIN lesions, p 5 3 mutations had at least one mutation that changes an amino acid, which may provide selection pressure for expansion. P C A with various grades of histologic differentiation is common. Previous studies using the fluorescence in situ hybridization (FISH) technique showed allelic loss of 8p in PIN and P C A lesions 32 and suggested a common "genetic history" for these proliferations. Also, F I S H studies revealed overexpression of c-myc. 33 Konishi et al. 34 reported different patterns of p 5 3 alterations among multifocal lesions of PCA. In cases 19 and 20 of our series, the direct sequencing of the PCRSSCP products from 4 independent foci, 2 PIN and 2 PCA, showed a different pattern of p 5 3 mutations, indicating each focus to be derived from different cell clones. The presence of different clones in the same prostatic lesions was also shown in another five cases (cases 6, 16, 17, 18, and 21). Multiclonality of prostatic precancerous and cancerous lesions is not surprising in the light of multistep carcinogenesis. The presence of precancerous lesions on the verge of becoming cancerous should be taken into account when treating patients with PCA. Our study 35 has shown that the HGPIN, like PCA, is sensitive to androgen deprivation therapy and is occasionally hard to recognize after hormone therapy, even on whole-mount prostatectomy specimens. The nontransition zone is known to be the dominant site for PCA and HGPIN. 36'37 Coexistence of P C A and H G P I N lesions in the nontransition zone was found in approximately 75% of PCA cases, 35 supporting the precancerous nature of HGPIN. Our study revealed that the frequency o f p 5 3 mutations in PCA lesions was significantly higher in the nontransition than in the transition zone. As for HGPIN, p 5 3 mutations in the nontransition zone were significantly more

31 A. Berner, G. Geitvik, E Karlsen, S. D. Fossa, J. M. Nesland, and A. L. Borresen, J. Pathol. 176, 299 (1995). 32j. Qian, D. G. Bostwick, S. Takahashi, T. J. Borell, J. E Herath, M. M. Lieber, and R. B. Jenkins, Cancer Res. 55, 5408 (1995). 33R. B. Jenkins, J. Qian, M. M. Lieber, and D. G. Bostwick, CancerRes. 57, 524 (1997). 34N. Konishi,Y. Hiasa, H. Matsuda,M. Tao, T. Tsuzuki, I. Hayashi, Y. Kitahori,T. Shiraishi,R. Yatani, J. Shimazaki, andJ. C. Lin, Am. J. Pathol. 147, 1112 (1995). 35M. Shin, H. Takayama,N. Nonomura, A. Wakatsuki,A. Okuyama, and K. Aozasa, Prostate 42, 81 (2000). 36j. E. McNeal, E. A. Redwine, E S. Freiha, and T. A. Stamey, Am. J. Surg. Pathol. 12, 897 (1988). 37 D. R. Greene, T. M. Wheeler, S. Egawa, J. K. Durra, andE T. A. Scardino,J. UroL 146,1069 (1991).

[9~8]

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frequent than in the transition zone. These findings suggest that p53 gene mutations play a role in the development of precancerous and cancerous lesions in the nontransition zone, but not in the transition zone. The distance between the PCA and HGPIN was reported to be frequently within 2 mm. 38 Bostwick and Brawer 1 reported that the frequency of appearance of HGPIN increased in cases with PCA compared to those without PCA. In our previous study 35 close association (distance within 2 mm) of HGPIN with PCA was more frequently found in the nontransition zone (63% of lesions) than in the transition zone (38% of lesions). These findings provide a basis for suggesting the precancerous nature of HGPIN, especially in the nontransition zone. Indeed, the frequency rate of p53 mutations in HGPIN lesions close to PCA (24% of lesions) was significantly higher than in those distant from PCA (>2 mm) (3% in total and none in the noncastrated cases). Our study using the laser capture microdissection method clearly showed the significant role of p53 gene mutations in the development of HGPIN and PCA in the nontransition zone with the sequential occurrence of HGP1N to PCA when these lesions were close to one another.

Fas Gene Mutations The death domain is necessary for the transduction of the apoptotic signa115'39'4°; therefore we examined mutations in exons 7, 8, and 332 bp of exon 9. DNA was subjected to first-round PCR of 10 cycles with the oligonucleofide primers followed by second PCR of 35 cycles with use of 0.1% of first-round PCR products as the template, denaturation for 30 sec at 95 °, annealing for 30 sec at variable temperatures, and extension for 30 sec at 72 ° (Table I) in a 9700 Applied Biosystems Thermocycler (Foster City, CA). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, CA) and were sequenced by the dideoxy chain termination method using the DNA sequencing kit (Applied Biosystems). The samples were analyzed with the Genetic Analyzer (ABI PRISM 310, Applied Biosystems). PCR products with suspicious mutations were cloned in the pCR 2.1-TOPO (Invitrogen), then sequenced to confirm whether the mutation exist. As shown in Table IV, 4 mutations of the Fas gene were detected in 4 HGPIN lesions from 4 cases. All mutations were point mutations; 3 missense and 1 nonsense mutation detected in exon 9, which encodes the death domain region of the Fas receptor. 14 Substitutions at codon 261 of the Fas cDNA sequence (GenBank

38 j. Qian and D. G. Bostwick, PathoL Res. Pract. 191, 860 (1995). 39 N. Itoh and S. Nagata, J. Biol. Chem. 268, 10932 (1993). 40 j. Cheng, O. Liu, and J. D. Mountz, J. lmmunol. 154, 1239 (1995).

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Laser Capture in Microscopy and Microdissection,

Laser Capture Microdissection

Laser Capture Microdissection: Methods and Protocols

Laser Capture Microdissection: Methods and Protocols (Methods in Molecular Biology)

Methods in Enzymology Vol 356: Laser Capture in Microscopy and Microdissection

Laser Capture Microdissection (Methods in Molecular Biology, v293)

Laser Capture Microdissection: Methods and Protocols, 2nd Edition (Methods in Molecular Biology Vol 755)

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Section cutting in Microscopy

Carbon capture and storage

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Light and video microscopy

Methods in Plant Electron Microscopy and Cytochemistry

Problems in laser physics

Opportunities in Laser Technology

Problems in Laser Physics

Molecules in laser fields

Laser age in optics

Light and video microscopy

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To Court, Capture and Conquer

Acoustic Microscopy

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Laser Capture in Microscopy and Microdissection,

Laser Capture Microdissection

Laser Capture Microdissection: Methods and Protocols

Laser Capture Microdissection: Methods and Protocols (Methods in Molecular Biology)

Methods in Enzymology Vol 356: Laser Capture in Microscopy and Microdissection

Laser Capture Microdissection (Methods in Molecular Biology, v293)

Laser Capture Microdissection: Methods and Protocols, 2nd Edition (Methods in Molecular Biology Vol 755)

Capture

Capture

Capture

Capture

Capture

Capture

Capture

Capture

Section cutting in Microscopy

Carbon capture and storage

The Capture

Light and video microscopy

Methods in Plant Electron Microscopy and Cytochemistry

Problems in laser physics

Opportunities in Laser Technology

Problems in Laser Physics

Molecules in laser fields

Laser age in optics

Light and video microscopy

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