Biological Microarrays: Methods and Protocols (Methods in Molecular Biology, Vol. 671)

Methods

in

Molecular Biology™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK



For other titles published in this series, go to www.springer.com/series/7651

Biological Microarrays Methods and Protocols

Edited by

Ali Khademhosseini Department of Medicine, MIT Division of Health Sciences and Tech, Harvard Medical School, Cambridge, MA, USA

Kahp-Yang Suh Department of Mechanical and Aerospace E, Seoul National University, Kwanak-gu, Shinlim-dong 56-1-1, 151-742, Seoul, Korea, Republic of (South Korea) Gwanak-ro 599, Gwanak-gu, Seoul 151-742, Republic of Korea

Mohammed Zourob Biophage Pharma, Montreal, QC, Canada

Editors Prof. Dr. Ali Khademhosseini Department of Medicine MIT Division of Health Sciences and Tech Harvard Medical School Cambridge, MA USA [email protected]

Dr. Mohammed Zourob Biophage Pharma Montreal, QC Canada [email protected]

Kahp-Yang Suh Department of Mechanical and Aerospace E Seoul National University Kwanak-gu, Shinlim-dong 56-1-1, 151-742 Seoul, Korea, Republic of (South Korea) Gwanak-ro 599, Gwanak-gu, Seoul 151-742, Republic of Korea [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-934115-95-4 e-ISBN 978-1-59745-551-0 DOI 10.1007/978-1-59745-551-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010938723 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or ­dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, ­neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface Microarrays are spatially ordered arrays with ligands chemically immobilized in discrete spots on a solid matrix, usually a microscope slide. Microarrays are a high-throughput large-scale screening system enabling simultaneous identification of a large number of target molecules (up to several hundred thousand) that bind specifically to the immobilized ligands of the array. Microarrays represent a promising tool for clinical, environmental, and industrial microbiology since the technology allows relatively rapid screening and identification of large number of specific analytes or genetic determinants simultaneously. The successful use of microarrays requires attention to unique issues of experimental design and execution. This book provides an overview of the methodology and applications of biological microarrays in various areas of biological and biomedical research. This book presents a significant and up-to-date review of the various biological microarrays, recognition elements, their immobilization, characterization techniques by a panel of distinguished scientists. This work is a comprehensive approach to the biological microarrays area presenting a thorough knowledge of the subject and an effective integration of these biological entities on microarray surfaces in order to appropriately convey the state-of-the-art fundamentals and applications of the most innovative approaches. This book comprises of 18 chapters written by 50 researchers actively working in USA, Canada, Germany, Spain, Korea, China, and the UK. The authors were requested to adopt a pedagogical tone in order to accommodate the needs of novice researchers such as graduate students and post-doctoral scholars as well as of established researchers seeking new avenues. This has resulted in duplication of some material, which we have chosen to retain, because we know that many readers will pick only a specific chapter to read at a certain time. We have divided this book into two major sections. The first part (Chaps. 1–9) comprises nine chapters, which are devoted to the application of biological microarrays including DNA/RNA, apatmer, proteins, tissues, oligonucleotides, carbohydrates, biomaterials, cells, bacteria, and virus microarrays. The second part (Chaps. 10–18) describes in detail the different techniques used for generating the microarray platforms. The second part divided into four subsections including photolithography (microfluidic-based approaches and cells and proteins patterns using photolithography), bioprinting (microspotters, microprinting), soft lithography (microcontact, micromolding, microstructure surface based on chemical vapor deposition, permeability of microvascular tubes), and microarray bioinformatics. It covers the theory behind each technique and delivers a detailed state-ofthe-art review for all the new technologies used. This book is intended to be a primary source both on fundamental and practical information of where the biological microarray area is now and where it is headed in the future. We anticipate that the book will be helpful to academics, practitioners and professionals working in various fields to name a few biologist, biotechnologists, biochemists, analytical chemists, biomedical, physical, microsystems engineering, nanotechnology, medicine, food, bioterrorism and security as well as allied health, health care, and surveillance. Since

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the fundamentals were also reviewed, we believe that the book will appeal to advanced undergraduate and graduate students who study in areas related to biological microarrays and biosensors. We gratefully acknowledge all authors for their participation and contributions, which made this book a reality. We give many thanks to Prof. John M. Walker for his guidance and patience. Last, but not least, we thank our families for their patience and enthusiastic support of this project.

Cambridge, MA Seoul, Korea Montreal, QC

Ali Khademhosseini Kahp-Yang Suh Mohammed Zourob

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v ix

Part I Application of Biological Microarray   1 RNA and DNA Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stuart C. Sealfon and Tearina T. Chu   2 Aptamer Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eva Baldrich   3 Oligonucleotide Microarrays for Identification of Microbial Pathogens and Detection of Their Virulence-Associated or Drug-Resistance Determinants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dmitriy V. Volokhov, Hyesuk Kong, Keith Herold, Vladimir E. Chizhikov, and Avraham Rasooly   4 Protein Microarrays Printed from DNA Microarrays . . . . . . . . . . . . . . . . . . . . . . Oda Stoevesandt, Mingyue He, and Michael J. Taussig   5 Lithographically Defined Two- and Three-Dimensional Tissue Microarrays . . . . . Esther W. Gomez and Celeste M. Nelson   6 Ratiometric Lectin Microarray Analysis of the Mammalian Cell Surface Glycome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ku-Lung Hsu, Kanoelani Pilobello, Lakshmipriya Krishnamoorthy, and Lara K. Mahal   7 Cell Microarrays Based on Hydrogel Microstructures for the Application to Cell-Based Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Won-Gun Koh   8 Fabrication of Bacteria and Virus Microarrays Based on Polymeric Capillary Force Lithography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pil J. Yoo   9 3D Polymer Scaffold Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carl G. Simon Jr., Yanyin Yang, Shauna M. Dorsey, Murugan Ramalingam, and Kaushik Chatterjee

3 35

55

95 107

117

133

147 161

Part II  Methods for Microarray Generation 10 PDMS Microfluidic Capillary Systems for Patterning Proteins on Surfaces and Performing Miniaturized Immunoassays . . . . . . . . . . . . . . . . . . . 177 Mateu Pla-Roca and David Juncker 11 Merging Photolithography and Robotic Protein Printing to Create Cellular Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Ji Youn Lee and Alexander Revzin

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12 Generation of Protein and Cell Microarrays on Functionalized Surfaces . . . . . . . . Yoo Seong Choi and Chang-Soo Lee 13 Microprinting of Liver Micro-organ for Drug Metabolism Study . . . . . . . . . . . . . Robert C. Chang, Kamal Emami, Antony Jeevarajan, Honglu Wu, and Wei Sun 14 Microcontact Printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yunyan Xie and Xingyu Jiang 15 Micromolding for the Fabrication of Biological Microarrays . . . . . . . . . . . . . . . . . Ashley L. Galloway, Andrew Murphy, Jason P. Rolland, Kevin P. Herlihy, Robby A. Petros, Mary E. Napier, and Joseph M. DeSimone 16 Progress Report on Microstructured Surfaces Based on Chemical Vapor Deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yaseen Elkasabi and Joerg Lahann 17 Methods for Forming Human Microvascular Tubes In Vitro and Measuring Their Macromolecular Permeability . . . . . . . . . . . . . . . . . . . . . . . Gavrielle M. Price and Joe Tien 18 Microarray Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robert P. Loewe and Peter J. Nelson

207 219

239 249

261

281 295

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

Contributors Eva Baldrich  •  Instituto de Microelectrónica de Barcelona (IMB-CNM), Barcelona, Spain Robert C. Chang  •  Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA, USA Kaushik Chatterjee  •  Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD, USA Vladimir E. Chizhikov  •  Center for Biologics Evaluation and Research, Food and Drug Administration, Kensington, MD, USA Yoo Seong Choi  •  Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea Tearina T. Chu  •  Departments of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY, USA Joseph M. DeSimone  •  North Carolina State University, Raleigh, NC, USA Shauna M. Dorsey  •  Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD, USA Yaseen Elkasabi  •  Material Science and Engineering, University of Michigan, Ann Arbor, MI, USA Kamal Emami  •  Radiation Physics Laboratory, NASA Johnson Space Center, Houston, TX, USA Ashley L. Galloway  •  Liquidia Technologies Research, Triangle Park, NC, USA Esther W. Gomez  •  Departments of Chemical Engineering and Molecular Biology, Princeton University, Princeton, NJ, USA Mingyue He  •  The Babraham Institute, Cambridge, UK Kevin P. Herlihy  •  Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Keith Herold  •  Department of Bioengineering, University of Maryland, College Park, MD, USA Ku-Lung Hsu  •  Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX, USA Antony Jeevarajan  •  Radiation Physics Laboratory, NASA Johnson Space Center, Houston, TX, USA Xingyu Jiang  •  National Center for NanoScience & Technology, Beijing, China David Junker  •  Bio-Medical Engineering Department, McGill University, Montreal, QC, Canada Won-Gun Koh  •  Department of Chemical and Biological Engineering, Yonsei University, Seoul, Korea Hyesuk Kong  •  Center for Biologics Evaluation and Research, Food and Drug Administration, Kensington, MD, USA

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Contributors

Lakshmipriya Krishnamoorthy  •  Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA Joerg Lahann  •  University of Michigan, Ann Arbor, MI, USA Chang-Soo Lee  •  Department of Chemical and Biological Engineering, Chungnam National University, Daejeon, Korea Ji Youn Lee  •  Department of Biomedical Engineering, University of California, Davis, CA, USA Robert P. Loewe  •  Medical Policlinic, Ludwig Maximillians, University of Munich, Munich, Germany Lara K. Mahal  •  Chemistry and Biochemistry Department, The University of Texas at Austin, Austin, TX, USA Andrew Murphy  •  Liquidia Technologies Research, Triangle Park, NC, USA Mary E. Napier  •  Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill NC, USA Celeste M. Nelson  •  Department of Chemical Engineering, Princeton University, Princeton, NJ, USA Peter J. Nelson  •  Medical Policlinic, Ludwig Maximillians, University of Munich, Munich, Germany Robby A. Petros  •  Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Kanoelani Pilobello  •  Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA Gavrielle M. Price  •  Department of Biomedical Engineering, Boston University, Boston, MA, USA Murugan Ramalingam  •  Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD, USA Avraham Rasooly  •  National Institutes of Health, National Cancer Institute, FDA, Bethesda, MD, USA Alexander Revzın  •  Department of Biomedical Engineering, University of California, Davis, CA, USA Jason P. Rolland  •  Liquidia Technologies Research, Triangle Park, NC, USA Stuart C. Sealfon  •  Neurology, Mount Sinai School of Medicine, New York, NY, USA Oda Stoevesandt  •  Babraham Bioscience Technologies, Cambridge, UK Wei Sun  •  Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA, USA Joe Tien  •  Department of Biomedical Engineering, Boston University, Boston, MA, USA Dmitriy V. Volokhov  •  Center for Biologics Evaluation and Research, Food and Drug Administration, Kensington, MD, USA Honglu Wu  •  Radiation Physics Laboratory, NASA Johnson Space Center, Houston, TX, USA Yunyan Xie  •  National Center for NanoScience & Technology, Beijing, China Yanyin Yang  •  Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD, USA

Contributors

xi

Pil J. Yoo  •  Chemical Engineering, Sungkyunkwan University, Seoul, Korea Michael J. Taussig  •  Babraham Bioscience Technologies, Cambridge,UK Carl G.Simon Jr  •  Polymers Division,National Institute of Standards and Technology, Gaithersburg, MD, USA Mateu Pla-Roca  •  Bio-Medical Engineering Department, McGill University, Montreal, QC, Canada



Part I Application of Biological Microarray

Chapter 1 RNA and DNA Microarrays Stuart C. Sealfon and Tearina T. Chu Abstract The development of microarray technology has revolutionized RNA and deoxyribonucleic acid (DNA) research. In contrast with traditional biological assays, microarrays allow the simultaneous measurement of tens of thousands of messenger RNA (mRNA) transcripts for gene expression or of genomic DNA fragments for copy number variation analysis. Over the past decade, genome-wide RNA or DNA microarray analysis has become an essential component of biology and biomedical research. The successful use of microarrays requires attention to unique issues of experimental design and execution. This chapter provides an overview of the methodology and applications of RNA and DNA microarrays in various areas of biological research. Key words: RNA, DNA, Expression, Comparative genomic hybridization, cDNA, BAC, Microarray, Copy number variation, Transcripts

1. Introduction Deoxyribonucleic acid (DNA) carries the hereditary information content of the genome, which is organized into discrete functional genes that regulate and encode individual RNAs. Genome size varies in organisms ranging from bacteria containing 1–5 million bases and 1,000–3,000 genes (1) to humans containing three billion bases and 20,000–25,000 protein-coding genes. Genes encoding protein are dynamically regulated and produce messenger RNA (mRNA) that are translated into protein. The human genome also contains thousands of nonprotein encoding RNA genes and large areas of regulatory and noncoding sequence (2). Measuring mRNAs indicate the level of gene activity and provide a snapshot of the biosynthetic state of the cell or tissue. The expression level of each gene can be influenced by a combination of genetic or environmental factors. The genetic factors include Ali Khademhosseini et al. (eds.), Biological Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 671, DOI 10.1007/978-1-59745-551-0_1, © Springer Science+Business Media, LLC 2011

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DNA polymorphisms in the regulatory regions of genes (such as the promoter/enhancer regions), and variations in the number of copies of the gene [copy number variation (CNV)]. Environment factors such as temperature, stress, nutrition, or exercise can lead to changes in extracellular hormones or intracellular signaling molecules that influence the expression level of genes. The expression levels of the genes of a cell determine the cell type, developmental stage, cell functions, and/or pathological state. However, it must be noted that the measurement of mRNA levels provides an imperfect reflection of protein levels and activity. The concentration of a particular protein is controlled not only by the level of its mRNA, but also by the rate of mRNA translation into protein and of protein degradation. Other modifications of protein, such as phosphorylation, are also important determinants of activity. With these limitations in mind, measurement of global mRNA expression provides insight into the overall level of gene activity and protein expression. Many human diseases involve altered gene expression (3). Small genomic deletions and duplications (1 kb to 10 Mb) constitute up to 15% of all mutations underlying human monogenic diseases (4). Thus, the study of small regions of chromosomal variation provides insight into the pathogenesis of many diseases. Changes in gene expression can arise from polymorphisms, deletions, or insertions in protein coding or regulatory sequences of DNA. Changes in gene expression can also arise from altered regulation of mRNA production in response to various signaling mechanisms or stimuli. In contrast with classical Mendelian genetics involving hereditable defects of a single gene locus, many diseases are polygenetic and have clusters of genes that may contribute to the pathological state (5–11). Microarray techniques that allow detection of small regions of DNA deletions or duplications play an important role in mapping diseases with a complex hereditary etiology. Microarray technology was first introduced in 1995 by Patrick Brown and colleagues (12). The first microarray was generated using complementary DNAs (cDNA) derived from polymerase chain reaction (PCR) products. The array was printed using a home-made robot and was used to measure the gene expression patterns in parallel of 48 Arabidopsis thaliana genes. Advances in microarray technology and the decoding of the human genome (13–15) as well as the genome of many other species (16–20), now make it feasible to assay simultaneously the expression level of tens of thousands of mRNA transcripts. We use the term RNA microarrays to refer to arrays used to measure RNA levels, whereas DNA microarrays measure DNA sequence or levels. RNA microarrays have been widely used to identify regulated genes, pathways, or gene networks in a variety of cells and tissues when two or more related biological conditions are compared.

RNA and DNA Microarrays

5

These approaches provide insight into biological mechanisms or cellular programs such as cell cycle progression (21–23), embryonic development (24–26), cell fate determination (27, 28), hormone responsive gene regulation programs (29–31), and drug or disease model -mediated gene expression changes (32–35). Microarrays have also been widely to define disease-associated gene regulation, gene expression patterns in disease subtypes, and gene biomarkers of various disease states such as cancers (36–40), infectious diseases (41–43), inflammatory disease (44, 45), neurological diseases (46–48), and psychiatric disorders (32, 49–51). In addition, microarray approach has been used in pharmacogenomic/ toxicogenomic studies for drug discovery, and for determining the mechanisms of therapeutic or side effects of specific drugs (35, 52–55). In the development of many human diseases, for example tumors, chromosomal damage leads to gain or loss of genomic material (4, 56–58). Comparative genomic hybridization (CGH) allows the study of the entire genome for variations in DNA copy number. Originally, metaphase chromosomes were used to represent the genome (59). This approach has limited resolution (~5–10  Mb for single copy gains and losses) and is technically difficult in requiring optimal chromosome metaphase spreads. Array-CGH (aCGH) circumvents some of these technical difficulties, and offers higher resolution (~1–100 kb intermarker spacing). This technique is useful for the detection of deletions or duplications of chromosomal regions or gene CNV in a comparison between individuals with altered disease states (60–62). Microarrays are important in cancer biology in identifying new tumor subtypes and prognostic groups. For example, breast cancer is a heterogeneous disease comprising many biological subtypes. After diagnosis, 30–40% of patients will develop metastases and die of the disease within 15 years. The selection of adjuvant chemotherapy is currently based on prognostic and predictive factors including age, tumor size, histological grade, hormone receptor status, Her2/neu status, menopausal status, and lymph node status (63, 64). Although these “classical” factors are effective based on general population statistics, they poorly predict the outcome for the individual patient because of the heterogeneity of this disease. Using RNA microarrays, Perou et al. was the first to divide breast carcinomas into distinct subtypes, based on the unique gene expression patterns of a subgroup of genes, called the “gene set” or “gene signature” (37). In a follow-up study, this gene set was used to predict the prognosis of 78 breast carcinoma patients (40). Using similar approaches, other workers have developed gene sets to predict the development of metastases and the prognosis of different groups (65–68). Microarrays have also been used to predict response to therapy (69–71). Using a DNA CGH microarray, Gonzalez-Neira et  al. identified a genetic

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classifier based on specific somatic genetic aberration of regions on chromosome 3p, 3q, and 5q to differentiate BRCA1 (breast cancer risk gene) mutation carriers from nonBRCA1 carriers of breast cancer patients (72). In a similar manner, array-CGH technology has been used in the characterization of different features of breast cancer including tumor stage classification and diagnostic/ prognostic subgroups of patients (for review see ref. 73). These studies raise the possibility that prediction of prognosis may be improved and that treatment for many diseases may be individualized based on gene analysis or gene signature patterns. The first diagnostic microarray to influence drug selection and dosage, the Roche AmpliChip Cytochrome P450 test, was approved by the FDA in 2004. Large-scale DNA or RNA analysis can be performed using microarrays generated by cloned libraries or synthetic oligonucleotides. The latter, nucleotide microarrays, will be discussed in a different section of this volume. In this chapter, we provide an introduction to the use, sample quality control, and sample preparation for cDNA-based RNA microarrays and BAC-based CGH DNA microarrays. The selection of reagents that are used in the materials and methods is based on authors’ preferences and experience. 1.1. RNA Microarray

Printing cDNA microarrays on glass slides was the first microarray technique developed and is still commonly used. cDNAs are amplified from individual clones in a library. Each cDNA fragment representing an individual gene of interest is immobilized on a glass slide that has been coated with DNA-binding chemicals such as amino silane or poly-l-lysine. These slide arrays can be printed as whole genome microarrays or with a focused selection of genes of interest. The two-color cDNA microarray assay is illustrated in Fig. 1. In a typical slide microarray experiment, the mRNAs from experimental samples to be compared (such as test vs. control) are reverse transcribed and are then labeled with two different detectable fluorescent markers (typically Cy3 vs. Cy5 or compatible Alexa dyes). When the amount of RNA in each sample is limited, such as assays from few or single cells, the RNAs may be subjected to an amplification procedure prior to fluorescent labeling. The two labeled samples are mixed and then hybridized to a microarray. After the excess of labeled probes is removed by washing, the intensity of each fluorophore at each array location is read using a laser scanner. Hybridization intensity is represented by the amount of fluorescent emission, which provides an estimate of the relative amount of each transcript present in the different samples. These arrays are printed using a library containing the sequences of interest. In a library, each bacteria clone carries a plasmid containing a unique sequence derived from the mRNA of a gene.

RNA and DNA Microarrays Test

RNA QC

Labeling QC

7

Control

or Total RNA RT & label or Amplify &

or cRNA

Fig.  1. Two-color RNA microarray assay. Test and control samples are reverse transcribed and labeled with different fluorophores. The labeled samples are competitively hybridized to the microarray that has been printed at each location with a different DNA sequence for a gene of interest. Determination of the relative fluorescence obtained at each array location, after normalization, reflects the relative levels of expression of each specific mRNA in the original samples. RT reverse transcription, QC quality control.

The library cDNAs, ranging from 500 bp to 2 kb, are amplified by PCR using the specific flanking primers of the gene, according to each clone library. The amplified cDNA fragments are purified, and the concentration determined. The end product of each clone is quality controlled by gel electrophoresis and printed using a specialized printing robot. 1.1.1. RNA QC

A slide RNA microarray offers a high level of flexibility in terms of labeling choices. The starting amount of RNA plays a significant role in influencing the choice of method used for target production, since it determines the level of amplification required. Three examples of labeling protocols are included in this section. Regardless of which method is chosen, the test and control samples must be properly matched to minimize the background variation.

1.2. DNA Microarray

CGH allows partial or entire genome analysis for variations in DNA copy number. In a CGH DNA microarray, artificial chromosomes from bacteria (BAC) containing known genomic

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fragments are immobilized on a glass surface and hybridized with a mixture of different fluorescence-labeled test and control DNAs. The ratio of the fluorescent intensities of the fluorophores is measured for each feature on the array. This ratio provides a relative measure of the difference in gene copy number between the samples. This technique is useful for a comparison between individuals with altered disease states in order to detect tissue-specific deletions or duplications of chromosomal regions. BAC genomic libraries can be purchased or custom made by an industrial provider or academic core facility, e.g., National Human Genome Research Institute, Wellcome Trust Sanger institute, Children’s Hospital Oakland Research Institute, Roswell Park Cancer Center, Clemson University Genomics Institute, Arizona Genomics Institute, Arabidopsis Biological Resource Center (ABRC) at Ohio State University, etc. The detailed protocol for preparation of the BAC DNAs is usually provided with the source library obtained, and the fabrication of a microarray slide is performed in an array printing service center. Briefly, BAC clones are streaked on LB-agar plates containing the appropriate antibiotic and grown overnight at 37°C. A single colony is inoculated in TB media containing the appropriate antibiotic and placed in a shaking incubator at 37°C for 16 h. From this culture, DNA is isolated and amplified through ligation-mediated PCR where a genomic DNA clone is digested with a restriction enzyme and a universal primer adaptor is ligated to serve as a priming site for PCR amplification. The amplified genomic DNA fragments are purified, the concentrations are determined and normalized, and then they are used for the fabrication of array slides for CGH. 1.2.1. DNA QC

Good quality of genomic DNA generally increases the sensitivity and accuracy of the array CGH assay. The DNA must be pure and free of contaminants, especially other sources of genomic DNA. Genomic DNA can be extracted from various sources, e.g., blood, buccal cells, cultured cells, tissue, and paraffin-embedded tissue. Many commercial kits targeting a particular sample source produce high-quality genomic DNA. Methods that include boiling or strong denaturants that may generate single-stranded DNA are not suitable to use. The purity of the DNA can be determined by the 260/280 spectrophotometer absorbance ratio. Ratio of 1.8 in a 10-mM Tris-HCl buffer typically represents pure DNA, whereas lower value for protein contamination and higher value for RNA contamination or degraded samples. The approximate average size of genomic DNA can be viewed on a 1% agarose gel. High-quality genomic DNA will run as a major peak at approximately 10–20 kb on the gel. Whole genome amplification (WGA) was developed in 1992 (74, 75) as a way to increase the amount of DNA from limited samples such as forensics and genetic disease research.

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Various WGA techniques have been developed. One approach, multiple displacement amplification (MDA) provides unbiased and accurate amplification of whole genomes (76, 77). This method utilizes Phi29 DNA polymerase, producing micrograms of high-molecular weight DNA fragments from as little as 10 ng of starting DNA. The end products are suitable for array CGH assay.

2. Materials 2.1. RNA QC 2.1.1. Direct Labeling

See Note 1 for preparing the mixture. 1. Oligo-dT18. 2. Superscript II. 3. 100 mM dNTP set. 4. Cy3- or Cy5- dUTP. 5. RNAsin. 6. RNAse H. 7. RNAseOne. 8. Minelute Cleanup Kit.

2.1.1.1. Stock Solutions and Master Mixtures

10× low dTTP dNTPs stock solution: Reagents

Amount

dGTP (100 mM)

  25 ml

dATP (100 mM)

  25 ml

dCTP (100 mM)

  25 ml

dTTP (100 mM)

  10 ml

DEPC-water

415 ml

Total

500 ml

Master Mix for making fluorescent cDNA target: Reagents

Amount

5× First strand buffer

6 ml

0.1 M DTT

3 ml

10× Low dTTP dNTP mix

0.8 ml

Cy-3 or Cy-5 dUTP (1 mM)

2 ml

RNAsin

1 ml

Total

12.8 ml

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2.1.2. Indirect Labeling

1. Oligo-dT18. 2. Superscript II. 3. 100 mM dNTP set. 4. aa-dUTP. 5. Sodium bicarbonate. 6. CyDye PostLabeling Reactive Dye Pack. 7. K2HPO4. 8. KH2PO4. 9. 0.5 M EDTA. 10. NaOH. 11. HCl. 12. MinElute cleanup kit. 13. NaAcetate. 14. DMSO.

2.1.2.1. Stock Solutions and Master Mixtures

Phosphate buffers (1  M Potassium phosphate, pH 8.5): Check pH with pH paper. Reagents

Amount

1 M K2HPO4

9.5 ml

1 M KH2PO4

0.5 ml

Total

10 ml

Phosphate wash buffer (5 mM KPO4, pH 8.5, 80% EtOH): Reagents

Amount

1 M Phosphate buffer, pH 8.5

0.5 ml

Water

15.25 ml

95% EtOH (alcohol)

84.25 ml

Total

100 ml

Phosphate elution buffer: Reagents 1 M Phosphate buffer, pH 8.5

Amount 4 ml

Water

996 ml

Total

1,000 ml

RNA and DNA Microarrays

11

100 mM Amino-allyl dUTP: Store in −20°C Reagents

Amount

aa-dUTP

1 mg

Water

19.1 ml

50× Labeling Mix (2:3 aa-dUTP:dTTP): Store in −20°C Reagents

Amount

dGTP (100 mM)

5 ml

dATP (100 mM)

5 ml

dCTP (100 mM)

5 ml

dTTP (100 mM)

3 ml

aa-dUTP (100 mM)

2 ml

Total

20 ml

0.3 M Sodium bicarbonate, pH 9.0: Check pH with pH paper. Use for 1 day only. Reagents

Amount

Sodium bicarbonate

1 g

dH2O

40 ml

NaOH (10 N)

180 ml

aa-dUTP-labeled cDNA target:

2.1.3. Small Sample Labeling

Reagents

Amount

5× First strand buffer

6 ml

0.1 M DTT

3 ml

50× Aminoallyl-dNTP mix

0.6 ml

Total

9.6 ml

1. Low input RNA amplification kit, see Note 2. 2. RNeasy MinElute Cleanup kit. 3. 50 mM aa-UTP. 4. 100 mM NTP set. 5. Sodium bicarbonate. 6. CyDye PostLabeling Reactive Dye Pack. 7. Hydroxylamine. 8. Fragmentation buffer.

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2.1.3.1. Stock Solutions and Master Mixtures

Low UTP NTP mixture: Aliquot 100  ml per tube and store at −80°C. Reagents

Amount

100 mM ATP

100 ml

100 mM GTP

100 ml

100 mM CTP

100 ml

100 mM UTP

  75 ml

DEPC-water

  25 ml

Total

400 ml

25  mM aa-UTP mixture: Aliquot 50  ml per tube and store at −80°C. Reagents

Amount

50 mM aa-UTP

100 ml

DEPC-water

100 ml

Total

200 ml

0.3 M Sodium bicarbonate at pH 9.0: Check pH with pH paper. Use for 1 day only. Reagents

Amount

Sodium bicarbonate

1 g

dH2O

40 ml

NaOH (10 N)

180 ml

cDNA Master Mix: Reagents

Amount

5× First strand buffer

4 ml

0.1 M DTT

2 ml

10 mM dNTP

1 ml

MMLV RT

1 ml

RNase OUT

0.5 ml

Total

8.5 ml

RNA and DNA Microarrays

In vitro transcription Master Mix:

2.1.4. Hybridization and Scanning (see Note 4)

Reagents

Amount

Water

5.7 ml

4× Transcription buffer

20 ml

0.1 M DTT

6 ml

Low UTP NTP Mix

16 ml

50% PEG

6.4 ml

RNAse OUT

0.5 ml

Inorganic pyrophosphatase

0.6 ml

aa-UTP (25 mM)

4 ml

T7 RNA polymerase

0.8 ml

Total

60 ml

1. Human or mouse Cot-1 DNA. 2. Poly(dA). 3. Transfer RNA (tRNA). 4. Formamide. 5. Succinic anhydride. 6. n-Methyl-pyrrilidinone. 7. NaBorate. 8. 50× Denhardt’s solution. 9. 20× SSPE. 10. 20× SSC. 11. SDS. 12. SS salmon sperm DNA. 13. Raised-edge coverslip. 14. Microarray hybridization cassette.

2.1.4.1. Stock Solutions

20× Blocking mixture: Reagents

Amount

Poly(dA)

40 mg

tRNA

80 mg

Human or mouse Cot-1 DNA 2,000 mg

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Sealfon and Chu

Add 1/10 volume of 3 M NaAcetate, pH 5.2, then precipitate the content with 2.5 × volume of 100% ethanol. Wash the pellet 1× with 70% ethanol and air-dry. Resuspend in 20-ml filtered Mili-Q water. Hybridization solution: Reagents

Amount

Formamide

35 ml

20× SSPE

20 ml

20% SDS

2.5 ml

50× Denhardt’s solution

5 ml

Water

37.5 ml

Total

100 ml

Prehybridization solution: Reagents

Amount

Formamide

  35 ml

20× SSPE

  20 ml

10% SDS

   5 ml

50× Denhardt’s solution

   5 ml

SS salmon sperm DNA (10 mg/ml)

   2 ml

Water

  33 ml

Total

100 ml

2.2. DNA QC

1. REPLIg Mini Kit.

2.2.1. Materials for Genomic DNA Amplification

2. Mini Quick Spin Column.

2.2.1.1. Stock Solutions and Master Mixtures (see Note 5)

Buffer D1 (sufficient for 15 reactions): Reagents

Amount

Reconstituted DLB buffer

  5 ml

Nuclease-free water

35 ml

Total

40 ml

Buffer N1 (sufficient for 15 reactions): Reagents

Amount

Stop solution

  8 ml

Nuclease-free water

72 ml

Total

80 ml

RNA and DNA Microarrays

15

Master Mix for amplification: Add the master mix components in the order listed in the table below. After addition of water and reaction buffer, briefly vortex and centrifuge the mixture before the addition of the DNA polymerase. The master mix should be kept on ice and used immediately upon the addition of the DNA polymerase.

2.2.2. Direct Labeling

Reagents

Amount

Nuclease-free water

10 ml

Reaction buffer

29 ml

DNA polymerase

  1 ml

Total

40 ml

1. BioPrime Array CGH Genomic DNA Labeling module. 2. 100 mM dNTP set. 3. 1 M Tris–HCl, pH 8.0. 4. 0.5 M EDTA, pH 8.0. 5. 1 mM Cy3 or Cy5-labeled dCTP. 6. Microcon YM 30.

2.2.2.1. Stock Solutions and Master Mixtures

10× dNTP Mix (0.5  mM dCTP, 2  mM dATP, 2  mM dGTP, 2 mM dTTP in TE buffer): Reagents

Amount

dGTP (100 mM)

4 ml

dATP (100 mM)

4 ml

dTTP (100 mM)

4 ml

dCTP (100 mM)

1 ml

1 M Tris–HCl, pH 8.0

2 ml

0.5 M EDTA, pH 8.0

0.4 ml

DEPC-water

184.6 ml

Total

200 ml

Master Mix for DNA labeling: Add the component in order listed. Reagents

Amount

10× dNTP

10 ml

Cy3 or Cy5-labeled dCTP

  4 ml

Klenow

  2 ml

Total

16 ml

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Sealfon and Chu

2.2.3. Hybridization and Scanning

1. 3 M NaAcetate, pH 5.2. 2. Ethanol, 100%, 80%. 3. Yeast RNA. 4. Herring sperm DNA. 5. Cot-1 DNA. 6. TE buffer, pH 8.0. 7. Formamide. 8. Dextran sulphate. 9. 10% Tween-20. 10. SSC. 11. 1 M Tris buffer pH 7.4. 12. Raised-edge coverslip. 13. Microarray hybridization cassette.

2.2.3.1. Stock Solutions and Master Mixtures

Reagents

Amount

Formamide

500 ml

Dextran sulphate

100 mg

Tween-20

1 ml

20× SSC

100 ml

1 M Tris buffer, pH 7.4

10 ml

Nuclease-free water

~389 ml

Total

1 ml

Pre-/hybridization buffer (50% formamide, 10% dextran sulphate, 0.1% Tween-20, 2× SSC, 10 mM Tris–HCl, pH 7.4).

3. Methods 3.1. RNA QC

Accurate measurement of transcripts requires RNA samples that are free of degradation, which can differentially affect individual sequences. The quality of the total RNA should be verified by two methods – spectrophotometer-based assay and visualization of the ribosomal RNA (rRNA). The 260/280 spectrophotometer absorbance ratio is the simplest test to assess RNA quality. Ratios between 1.9 and 2.1 in a 10-mM Tris-based buffer typically represent high quality, pure RNA. Values considerably below this range suggest DNA, protein, or chemical contamination. Values greater than this range suggest the presence of degraded RNAs. A more sensitive method to assess the integrity of RNA is to

RNA and DNA Microarrays

a

17

c RIN=9.6 18S rRNA

RIN=5.5

28S rRNA

Leading marker

b

d RIN=8.3

RIN=2.8

Fig. 2. Representative Bioanalyzer electropherogram showing RNA samples of varying quality. RIN: RNA integrity number, ranging from 10 to 0 for the best to worst RNA integrity. RNAs in panels (c) or (d) are not suitable for RNA microarray assay.

visualize the rRNA component of total RNA. This can be achieved by performing an RNA gel electrophoresis; however, the process is tedious and requires micrograms levels of RNA. The Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA), a microfluidics-based platform, is a satisfactory way to assess RNA quality using small quantities of RNA. Representative Bioanalyzer readouts are depicted in Fig. 2. In addition to providing a “gel-like” image of the sample, this system derives an RNA integrity number or RIN that is useful in estimating the overall quality of total RNA samples. 3.1.1. Direct Labeling Methods

Reagents’ list and amounts are given in Subheading  2.1.1 to prepare Stock Solutions or Master Mixtures. 1. Resuspend 20–50 mg of total RNA or 1–2 mg of mRNA in DEPC-H2O to make the final volume of 13.2 ml. 2. Add 2 ml of oligo-dT18 (2 mg/ml). 3. Final volume: 15.2 ml. 4. Incubate at 65°C for 10 min. 5. Place on ice for at least 2 min.

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3.1.1.1. To Make Fluorescent cDNA Target

1. Add the mixed content, given in Subheading  2.1.1, to the annealed RNA mix. 2. Add 2 ml of superscript II into the mixture. 3. Final volume: 30 ml. 4. Incubate at 42°C for 1 h. 5. Add another 1 ml of superscript II. 6. Incubate for another 1 h. 7. Heat at 94°C for 2 min.

3.1.1.2. To Degrade RNA

1. Add 48 ml dH2O to each tube. 2. Add 9 ml 10× RNAse One buffer. 3. Add 2 ml RNAse One. 4. Incubate at 37°C for 10 min. 5. Heat 94°C for 1 min.

3.1.1.3. To Cleanup cDNA Targets by MinElute Column

1. Keep Cy5 and Cy3 separate for MinElute cleanup to measure CyDye incorporation, if desired. 2. Add 9 ml 3 M NaAcetate, pH 5.2 to the sample tube. 3. Add 495 ml binding buffer to each sample. 4. Assemble the MinElute column on the provided 2-ml collection tubes. 5. Load the entire mixture to a MinElute column. 6. Centrifuge for 1  min at 10,000  RCF. Discard the flowthrough and reuse the 2-ml tube. 7. Add 750 ml PE wash buffer to the column. 8. Centrifuge at 10,000  RCF for 1  min. Discard the flowthrough and reuse the 2-ml tube. 9. Repeat steps 7 and 8. 10. Centrifuge again at maximum speed for 1  min to remove residual EtOH. 11. Place column in a fresh 1.5-ml tube. Add 10 ml of water (pH 7.5) to elute. 12. Allow elution water to stand for at least 2  min before spinning. 13. Centrifuge at maximum speed for 1 min. Add 10 ml of water (pH 7.5) to elute. 14. Allow elution water to stand for at least 2  min before spinning. 15. Centrifuge at maximum speed for 1 min. 16. Proceed to “Analysis of Target Labeling Reaction by NanoDrop Spectrophotometer” in Subheading 3.1.3.7.

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19

17. Combine equal amount of the exp- and control-labeled cDNA targets. Bring the final volume to 18.5  ml. Reduce volume by speedvac if necessary. 3.1.2. Indirect Labeling Methods

Reagents’ list and amounts are given in Subheading  2.1.2 to prepare Stock Solutions or Master Mixtures.

3.1.2.1. To Anneal RNA

1. Resuspend 20–50 mg of total RNA or 1–2 mg of mRNA in DEPC-H2O to make the final volume to 16.4 ml. 2. Add 2 ml of oligo-dT18 (2 mg/ml). 3. Final volume: 18.4 ml. 4. Incubate at 65°C for 10 min. 5. Quick spin and place on ice for at least 2 min.

3.1.2.2. To Make aa-dUTP-Labeled cDNA Target

1. Add the mixed content given in Subheading  2.1.2 to the annealed RNA mix. 2. Add 2 ml superscript II into the mixture. 3. Final volume: 30 ml. 4. Incubate at 42°C for 2 h. 5. Add another 1 ml superscript II. 6. Incubate for another 1 h. 7. Add 1 ml 0.5 M EDTA, see Note 6.

3.1.2.3. RNA Hydrolysis

1. Heat mixture at 95°C for 3 min. 2. Quick spin and immediately place on ice for at least 2 min. 3. Add 15 ml 1 M NaOH. 4. Mix and incubate at 65°C for 15 min. 5. Quick spin and put the tube on ice. 6. Add 15 ml 1 M HCl. 7. Total volume: 62 ml.

3.1.2.4. Targets Purification (see Note 7)

1. Add 6 ml 3 M NaAcetate, pH 5.2 to each sample tube. 2. Add 340 ml binding buffer to each sample. 3. Assemble the MinElute column on the 2-ml collection tubes provided. 4. Load the entire mixture to a MinElute column. 5. Centrifuge for 1  min @ 10,000  RCF. Discard the flowthrough and reuse the 2-ml tube. 6. Add 750 ml phosphate wash buffer to the column. 7. Centrifuge at 10,000 RCF for 1 min. Discard the flow-through and reuse the 2-ml tube. 8. Repeat steps 6 and 7.

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Sealfon and Chu

9. Centrifuge again at maximum speed for 1  min to remove residual EtOH. 10. Place column in a fresh 1.5-ml tube. Add 10 ml of phosphate elution buffer to elute. 11. Allow elution buffer to stand for at least 2  min before spinning. 12. Centrifuge at maximum speed for 1  min. Add 10  ml phosphate elution buffer to elute. 13. Allow elution buffer to stand for at least 2 min before spinning. 14. Centrifuge at maximum speed for 1 min. 15. Dry sample completely in a speedvac. 3.1.2.5. Coupling aa-cDNA to Cy Dye Ester

1. Resuspend sample in 6 ml water. 2. Add 3 ml 0.3 M Na2CO3 buffer, pH 9.0. 3. Total volume: 9 ml. 4. Add 11 ml high-quality DMSO to one tube of Cy3 or Cy5 dye. 5. Vortex to mix thoroughly. Keep dye in the dark until ready to use (do not prepare dye >1 h before using). Make sure that no water gets into the dye/DMSO mix at any point. 6. Transfer the dye mix to sample tube. 7. Total volume: 20 ml. 8. Incubate at RT in the dark for 1 h.

3.1.2.6. Target Purification

1. To the tube, add 70 ml H2O and 10 ml 3 M NaOAc, pH 5.2. 2. Add 500 ml binding buffer. 3. Assemble the MinElute column on the provided 2-ml collection tubes. 4. Load the entire mixture to a MinElute column. 5. Centrifuge for 1  min at 10,000  RCF. Discard the flowthrough and reuse the 2-ml tube. 6. Add 750 ml PE buffer to the column. 7. Centrifuge at 10,000  RCF for 1  min. Discard the flowthrough and reuse the 2-ml tube. 8. Repeat steps 6 and 7. 9. Centrifuge again at maximum speed for 1  min to remove residual EtOH. 10. Place column in a fresh 1.5-ml tube. Add 10  ml of water (pH 7.5) to elute. 11. Allow elution buffer to stand for at least 2  min before spinning. 12. Centrifuge at maximum speed for 1 min. Add 10 ml of water (pH 7.5) to elute.

RNA and DNA Microarrays

21

13. Allow elution water to stand for at least 2 min before spinning. 14. Centrifuge at maximum speed for 1 min. 15. Put sample on ice and in the dark. 16. Proceed to “Analysis of Target Labeling Reaction by NanoDrop Spectrophotometer” in Subheading 3.1.3.7, if desired. 17. Combine the test- and control-labeled cDNA targets. Bring the final volume to 18.5  ml. Reduce volume by speedvac if necessary. 3.1.3. Small Sample Labeling Methods

3.1.3.1. RNA Annealing

Reagents’ list and amounts are given in Subheading  2.1.3 to prepare Stock Solutions or Master Mixtures. Preset a PCR program with lid heat off as follows: ●

65°C 10 min

●

4°C 5 min

●

4°C pause

●

40°C 2 h

●

65°C 15 min

●

4°C 5 min

●

4°C hold

1. Use 50 ng to 5 mg of total RNA per reaction. If possible, start with 2 mg of total RNA and make the final volume to 6.5 ml. 2. Add 5 ml T7 promoter primer. 3. Place the tube in a preprogrammed PCR machine. 4. Incubate at 65°C for 10 min, 4°C for 5 min and pause.

3.1.3.2. cDNA Synthesis

1. Prewarm 5× first strand buffer at 80°C for 3–4 min. Quick spin the tube and keep at RT until use. 2. To each sample tube, add 8.5 ml of cDNA Master Mix. 3. Incubate samples at 40°C for 2 h, 65°C for 15 min, 4°C for 5 min, then 4°C hold (see Note 8).

3.1.3.3. In Vitro Transcription

1. Prewarm the 50% PEG solution at 40°C for 1 min. 2. In a separate tube, prepare a master mix, given in Subheading 2.1.3 at RT immediately prior to use. 3. Add 60 ml of transcription master mix to each sample tube. 4. Total volume: 80 ml. 5. Place tubes on PCR machine. Run program 6. 40°C 2 h 7. 4°C hold 8. Add 20 ml RNase-free water to each sample tube to make a total volume of 100 ml.

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3.1.3.4. cRNA Purification with RNeasy MinElute Column

See Note 9 for cRNA purification. 1. Prewarm RNase-free water at 50°C for at least 10 min. 2. Add 350 ml Buffer RLT, and mix thoroughly. 3. Add 250 ml ethanol (96–100%) to the mixture, and mix thoroughly by pipetting. Do not centrifuge. Continue immediately with step 4. 4. Apply the sample (700 ml) to an RNeasy MinElute column placed in a 2-ml collection tube (supplied). Close the tube gently, and centrifuge for 30 s at ³10,000 × g. 5. Discard the flow-through and collection tube. 6. Transfer the RNeasy column into a new 2-ml collection tube (supplied). Pipet 500  ml Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 1  min at ³10,000 × g to wash the column. Discard the flow-through. Reuse the collection tube in step 6. 7. Add 500  ml 80% ethanol to the RNeasy column. Close the tube gently, and centrifuge for 1 min at ³10,000 × g. Discard the flow-through. Reuse the collection tube and centrifuge for additional 2 min. 8. To elute, transfer the RNeasy column to a new 1.5-ml collection tube. Pipet preheated 10  ml RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube gently, let sit at room temperature for 1  min, and centrifuge for 1 min at ³10,000 × g to elute. 9. Repeat step 8 once. 10. Quantitate cRNA yield by spectrophotometer.

3.1.3.5. Cy Dye Coupling Reaction

1. Add 11  ml of high-quality DMSO to each dye tube. Mix thoroughly and keep in dark. 2. Use 5  mg of aa-modified cRNA and vacuum dry (not to complete dryness). 3. Adjust volume to 6 ml. 4. Add 3 ml 0.3 M sodium bicarbonate buffer, pH 9.0 to sample tube. 5. Transfer the Cy-DMSO dye solution to sample tube and mix well. 6. Total volume: 20 ml. 7. Incubate in the dark at RT for 1 h. 8. Add 4.5 ml 4 M hydroxylamine solution to the mixture and incubate for 15 min in the dark at RT. 9. Add 5.5 ml DEPC-water to the labeled cRNA. 10. Total volume: 30 ml.

RNA and DNA Microarrays 3.1.3.6. Labeled cRNA Purification with RNeasy MinElute Column (see Note 8)

23

1. Prewarm RNase-free water at 50°C for at least 10 min. 2. Add 105 ml Buffer RLT, and mix thoroughly. 3. Add 75 ml ethanol (96–100%) to the mixture, and mix thoroughly by pipetting. Do not centrifuge. Continue immediately with step 4. 4. Apply the sample mixture (210 ml) to an RNeasy MinElute column placed in a 2-ml collection tube (supplied). Close the tube gently, and centrifuge for 30 s at ³10,000 × g. 5. Discard the flow through and collection tube. 6. Transfer the RNeasy column into a new 2-ml collection tube (supplied). Pipet 500  ml Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 1  min at ³10,000 × g to wash the column. Discard the flow-through. Reuse the collection tube in step 6. 7. Add 500  ml 80% ethanol to the RNeasy column. Close the tube gently, and centrifuge for 1 min at ³10,000 × g. Discard the flow-through. Reuse the collection tube and centrifuge for additional 1 min. 8. To elute, transfer the RNeasy column to a new 1.5-ml collection tube. Pipet preheated 10  ml RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube gently; let it sit at room temperature for 1  min, and centrifuge for 1 min at ³10,000 × g to elute. 9. Repeat step 8 once. 10. Proceed to “Analysis of Target Labeling Reaction by NanoDrop Spectrophotometer” in Subheading  3.1.3.7 if desired. 11. Combine equal amount of the labeled cRNAs, approximately 100  pmol of Cy3 and 50  pmol Cy5 for each hybridization reaction. 12. Bring the volume to 20 ml with Nuclease-free water (or reduce volume in a speedvac if necessary). Do not dry completely. 13. Proceed to hybridization.

3.1.3.7. Analysis of Target Labeling Reaction by NanoDrop Spectrophotometer

1. Start the NanoDrop software. 2. Click the MicroArray tab. 3. Before initializing the instrument as requested by the software, clean the sample loading area with nuclease-free water. 4. Load 1.0 ml of nuclease-free water to initialize. Then, click OK. 5. Once the instrument has initialized, select RNA-40 (for cRNA), ssDNA-33 (for cDNA), or DNA-50 (for genomic DNA) as the Sample type (use the drop down menu) or according to your sample.

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Sealfon and Chu

6. Make sure the Recording button is selected. If not, click Recording so that the readings can be recorded, saved, and printed. 7. Blank the instrument by pipetting 1.0 ml of nuclease-free water or elution buffer (whatever the samples are in) and click Blank. 8. Clean the sample loading area with a laboratory wipe. Pipette 1.0 ml of the sample onto the instrument sample loading area. Type the sample name in the space provided and click Measure (see Note 10). 9. Similarly, measure the RNA, ssDNA, or DNA absorbance by clicking the NucleicAcid tab in the main menu. 10. Print the results. If printing the results is not possible, record the following values: ●

Cyanine 3 or cyanine 5 dye concentration (pmol/ml)

●

RNA, ssDNA, or DNA absorbance ratio (260/280 nm)

●

cRNA, ssDNA, or DNA concentration (ng/ml)

11. Determine the yield and specific activity of each reaction as follows: ●

●

●

Use the concentration of RNA or DNA (ng/ml) to determine the mg RNA or DNA yield as follows: (Concentration of RNA or DNA) × (elution volume)/1,000 = mg of RNA or DNA. Use the concentrations of RNA or DNA (ng/ml) and cyanine 3 or cyanine 5 (pmol/ml) to determine the specific activity as follows: (Concentration of Cy3 or Cy5)/ (Concentration of RNA or DNA) × 1,000 = pmol Cy3/mg RNA or DNA. Use the A260 and A550 (for Cy3) or A650 (for Cy5) to determine the base-to-dye ratio as follows: Base/dye for Cy3™ = [A260 × 150,000 (cm–1 M–1)]/(A550 × 6,600). Base/dye for Cy5™ = [A260 × 250,000 (cm–1 M–1)]/ (A650 × 6,600). The base-to-dye ratio should be 40–80 for both Cy3 and Cy5.

12. Examine the yield and specific activity results. 13. If the yield is