DNA Microarrays, Part A: Array Platforms and Wet-Bench Protocols (eBook)

Alan R. Kimmel, Brian Oliver (Herausgeber)

eBook Download: PDF

2011 | 1. Auflage
512 Seiten
Elsevier Science (Verlag)
978-0-08-046465-7 (ISBN)

Infos

Modern DNA microarray technologies have evolved over the past 25 years to the point where it is now possible to take many million measurements from a single experiment. These two volumes, Parts A & B in the Methods in Enzymology series provide methods that will shepard any molecular biologist through the process of planning, performing, and publishing microarray results.

Part A starts with an overview of a number of microarray platforms, both commercial and academically produced and includes wet bench protocols for performing traditional expression analysis and derivative techniques such as detection of transcription factor occupancy and chromatin status. Wet-bench protocols and troubleshooting techniques continue into Part B. These techniques are well rooted in traditional molecular biology and while they require traditional care, a researcher that can reproducibly generate beautiful Northern or Southern blots should have no difficulty generating beautiful array hybridizations.

Data management is a more recent problem for most biologists. The bulk of Part B provides a range of techniques for data handling. This includes critical issues, from normalization within and between arrays, to uploading your results to the public repositories for array data, and how to integrate data from multiple sources. There are chapters in Part B for both the debutant and the expert bioinformatician.

? Provides an overview of platforms
? Includes experimental design and wet bench protocols
? Presents statistical and data analysis methods, array databases, data visualization and meta analysis

Inhalt

Cover Page 1
Table of Contents 6
Contributors to Volume 410 10
Volumes in Series 14
Section I Array Platforms 38
Chapter 1: The Affymetrix GeneChipreg Platform: An Overview 40
Introduction 40
GeneChip Microarrays, a Flexible Platform 42
Array Manufacturing 42
Array Design 45
Target Preparation 50
GeneChip Instrument Components and Associated Assay Steps 54
Image and Data Analysis 55
Current Applications 56
Advancing the Future of Genomics 61
Acknowledgments 61
References 62
Chapter 2: The Agilent In Situ-Synthesized Microarray Platform 65
Introduction 65
Technology 66
Applications 67
Methods Descriptions 68
Array Design 68
Sample Isolation, Labeling, and Quality Control 69
Array Hybridization and Scanning 70
Data Extraction 71
Array Design 72
Process Description 72
Sample Isolation, Labeling, and Quality Control 74
Array Hybridization and Scanning 79
Data Extraction 84
References 90
Chapter 3: Illumina Universal Bead Arrays 94
Introduction 95
Material and Methods 95
Results and Discussion 102
Conclusion 107
Acknowledgments 108
References 108
Chapter 4: Microarray Oligonucleotide Probes 110
The Case for Oligonucleotide Probes 111
Considerations for Oligonucleotide Probe Design 112
Microarray Production and Hybridization Protocols 114
Practical Considerations in Probe Sequence Design, a Case Study 115
Employment and Postprocessing 118
Conclusion 120
Locations of Probe Sequence Target Regions: Discrimination of Highly Similar Targets 120
In Situ Synthesis vs Deposition of Presynthesized Oligonucleotides 122
Thermodynamic Modeling of Microarray Probe Hybridization 123
Outlook 130
Supplement 131
Acknowledgments 131
References 131
Chapter 5: Automated Liquid Handling and High-Throughput Preparation of Polymerase Chain Reaction-Amplified DNA for Microarray Fabrication 136
Introduction 136
Overview 137
Methods 145
Summary 156
Acknowledgments 157
References 157
Chapter 6: The Printing Process: Tips on Tips 158
Introduction 158
The Printing Process and Equipment 159
Printing Pin Technology Options 161
Critical Parameters and Troubleshooting 163
Quality Control Testing 168
Future Developments 170
Acknowledgment 171
References 172
Chapter 7: Making and Using Spotted DNA Microarrays in an Academic Core Laboratory 172
Introduction 173
Technologies and Services Provided by the Keck Microarray Resource 174
Generic Glass Slide Microarray Printing 178
Genomic Solutions OmniGrid 100 Microarrayer Print Settings 179
Materials and General Settings for cDNA and Oligonucleotide Printing on In-House PLL-Coated Slides 180
Quality Control Parameters for DNA Microarray Printing and Analysis Using Spotted Glass Slides 181
Labeling and Hybridization Protocols Employed by the Keck Microarray Resource 183
General Considerations Regarding RNA Samples, Labeling, and Hybridization Strategies 187
Indirect Amino-Allyl dUTP Target Labeling, Monofunctional Dye Conjugation, and cDNA and Oligonucleotide Array Hybridization Protocols 189
Genisphere 3DNA Dendrimer Labeling and Oligonucleotide Array Hybridization 192
Genicon/Invitrogen Resonance Light Scattering Using Gold and Silver Nanoparticle Labeling with HiLight Scanner Detection 196
Scanning and Analyzing Spotted Microarrays 199
Yale Microarray Database 199
Gene Expression Studies Using Keck Microarray Slides and/or Services 202
Acknowledgments 204
References 204
Chapter 8: Printing Your Own Inkjet Microarrays 205
Introduction 206
Description of Methods 214
Concluding Remarks 223
References 224
Chapter 9: Peptide Nucleic Acid Microarrays Made with (S,S)-trans-Cyclopentane-Constrained Peptide Nucleic Acids 226
Introduction 226
Experimental Section 228
General Procedure for Manual Solid-Phase Synthesis of Peptide Nucleic Acids 233
Procedure Used to Attach PNA to Surface 236
References 236
Section II Wet-Bench Protocols 238
Chapter 10: Optimizing Experiment and Analysis Parameters for Spotted Microarrays 240
Introduction 240
Factors Affecting the Quality of Experimental Data 241
Factors to Consider in Experimental Design and Analysis 244
Perspectives and Conclusions 255
Acknowledgments 257
References 257
Chapter 11: Sample Labeling: An Overview 259
Extraction of Nucleic Acids 260
Template Amplification 263
Labeling 266
Conclusions 270
References 271
Chapter 12: Genomic DNA as a General Cohybridization Standard for Ratiometric Microarrays 274
Introduction 275
Current Cohybridization Standards: RNA 277
DNA Standards 277
Evaluating Cohybridization Standards 278
Recovering Information on RNA Prevalence 278
Shelf Life and Quality Control for Genomic DNA Standards 279
Method 280
Equipment and Reagents 282
Freezing and Pulverization 283
Tissue Lysis 283
Protein Precipitation 284
Genomic DNA Precipitation and Resuspension 284
RNase Digestion 285
Sonication 285
Column Cleanup 285
Labeling Method I. Direct Incorporation of Cy-Labeled dCTP 286
Equipment and Reagents 287
Labeling 288
Cleanup 289
Prepare the Labeled Target for the Array 289
Fluorometric Quantification of Reaction Yield and Label Incorporation 290
Equipment and Reagents 290
Quantifying Yield and Incorporation Efficiency 290
Labeling Method II. Indirect Labeling via Incorporation of Amino-allyl dUTP 291
Equipment and Reagents 293
Protocol for Resuspension of Dry Cy Dye in DMSO 294
10x Aminoallyl-dUTP/dNTPS Labeling Mix 295
Denature Sonicated Genomic DNA Template with NaOH 295
Cleanup over Zymo Columns (either 5 or 25 mug Capacity) 295
Annealing to Random Primers 296
Labeling 296
Cleanup 297
Coupling to Monofunctional Reactive Dye 297
Qiagen PCR Column to Remove Enzyme and Uncoupled Dye from Sample 298
Final Cleanup over P30 Biogel Chromatography Column 298
Prepare the Labeled Target for the Array 299
Quantifying Yield and Incorporation Efficiency 299
Conclusions 310
Acknowledgments 313
References 313
Chapter 13: Analysis of Sequence Specificities of DNA-Binding Proteins with Protein Binding Microarrays 316
Introduction 316
Preparation of DNA Microarrays for Use in Protein Binding Microarray Experiments 320
Protein Binding Microarray Experiments 324
Analysis of Protein Binding Microarray Data 328
Conclusions 334
Acknowledgments 335
References 335
Chapter 14: Microarray Analysis of RNA Processing and Modification 337
Introduction 337
Microarrays for Measuring Noncoding RNA Processing and Modification 338
Microarrays for Covalent RNA Modification 344
Microarrays for Monitoring Alternative Splicing 344
Alternative Splicing Microarray Design 347
Conclusion 351
Acknowledgments 351
References 351
Chapter 15: Mapping the Distribution of Chromatin Proteins by ChIP on Chip 353
Introduction 354
Chromatin Immunoprecipitation 356
Chips for ChIPs 362
Labeling and Hybridization 366
Data Acquisition 368
Statistical Analysis of Data 370
Graphic Comparison of ChIP on Chip Data with Genome Annotations 374
Perspectives 375
Acknowledgments 375
References 376
Chapter 16: DamID: Mapping of In Vivo Protein-Genome Interactions Using Tethered DNA Adenine Methyltransferase 379
Introduction 379
Principle of DamID 380
Correcting for Untargeted Binding of Dam 381
Expression Levels of Dam and Dam Fusion Proteins 382
Comparison of DamID and ChIP 384
Materials 385
Methods 387
Acknowledgments 395
References 395
Chapter 17: Whole-Genome Genotyping 396
Introduction 397
Methods 405
Conclusions 411
Acknowledgments 412
References 412
Chapter 18: Mapping Drosophila Genomic Aberration Breakpoints with Comparative Genome Hybridization on Microarrays 414
Introduction 414
Materials and Methods 417
Acknowledgments 422
References 422
Chapter 19: Performing Quantitative Reverse-Transcribed Polymerase Chain Reaction Experiments 423
Introduction 424
Choice of the Fluorescence Format 425
Choice of the Enzymatic System 425
Amplification and Denaturation Curves 426
Determination of the Correct Annealing Temperature: Specificity 429
Checking the Amplification Yield: Efficiency 429
Relative Quantification 432
Multiplex Polymerase Chain Reaction 434
Proposed Reaction Medium for SYBR Green Q-PCR 434
Conclusion 435
References 436
Chapter 20: The Application of Tissue Microarrays in the Validation of Microarray Results 437
Introduction 437
Ethical Concerns in the Use of Tissue in Biomedical Research 439
Determining What Tissue to Analyze 440
Construction of a Tissue Microarray 441
Obtaining a Tissue Microarray from Other Sources 443
Confirmation, Validation, and Determining Experimental Approach 444
Immunohistochemical Assays for Validation 445
In Situ Assays 447
Collection and Interpretation of Data 448
Analysis of Data 449
Other Applications of TMAs Relating to Microarrays 450
Conclusions 451
References 451
Chapter 21: Mapping Histone Modifications by Nucleosome Immunoprecipitation 453
Introduction 453
Preparation of Core Particles: MNase Digestion of Minichromosomes 455
Immunoprecipitation of Nucleosomes 457
Radioactive End Labeling of Nucleosome Core Particle DNA 458
Analysis of Immunoprecipitated Nucleosomes 458
Alkali Denaturation of Immunoprecipitated, Labeled Core Particle DNA 462
Monomer Extension 462
Analysis of the Histone Modification Map 464
Concluding Remarks 465
Acknowledgments 466
References 466
Author Index 468
Subject Index 498

Produkt-Details

• Erscheint lt. Verlag: 19.8.2011
• Sprache: englisch
• Themenwelt: Informatik ► Weitere Themen ► Bioinformatik
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Technik
• ISBN-10: 0-08-046465-3 / 0080464653
• ISBN-13: 978-0-08-046465-7 / 9780080464657