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
Author(s): Alan R. Kimmel, Brian Oliver
Series: Methods in Enzymology 411 Part B
Edition: 1
Publisher: Academic Press
Year: 2006
Language: English
Pages: 446
23.pdf......Page 0
Introduction......Page 9
Array Manufacturing......Page 11
Array Design......Page 14
Array Designs for Gene Expression......Page 15
Array Design for DNA Analysis......Page 17
Target Preparation......Page 19
GeneChip Instrument Components and Associated Assay Steps......Page 23
Image and Data Analysis......Page 24
Current Applications......Page 25
Expression......Page 26
Genotyping......Page 28
Acknowledgments......Page 30
References......Page 31
Introduction......Page 35
Technology......Page 36
Applications......Page 37
Preferred/Supported Methods......Page 38
Preferred Methods......Page 39
Preferred Methods......Page 40
Other Published Methods......Page 41
Process Description......Page 42
Preferred/Supported Methods......Page 44
Cleanup of Digested DNA......Page 46
Genomic DNA Labeling......Page 47
Prewarming of Wash Solutions......Page 49
Hybridization......Page 50
Wash......Page 52
Scanning Using Agilent Scanner......Page 53
Preferred/Supported Methods......Page 54
Troubleshooting Guide......Page 57
References......Page 60
Illumina Universal Bead Arrays......Page 65
BeadArray Technology......Page 66
Array of Arrays Format......Page 68
GoldenGate Genotyping Assay......Page 69
Biotinylation of Genomic DNA (Step 1)......Page 70
PCR Amplification (Step 4)......Page 71
Automatic Genotype Scoring (Step 9)......Page 72
Genotyping Throughput......Page 73
Implementation of the Methylation Profiling Assay on the SNP Genotyping Platform......Page 75
High-Throughput Gene Expression Profiling on Universal Array Matrices......Page 76
Conclusion......Page 78
References......Page 79
Microarray Oligonucleotide Probes......Page 82
The Case for Oligonucleotide Probes......Page 83
Number of Probes per Target and Probe Lengths......Page 84
Microarray Production and Hybridization Protocols......Page 86
Practical Considerations in Probe Sequence Design, a Case Study......Page 87
Choice of Design Parameters, the Search for "Optimal" Probes......Page 89
Employment and Postprocessing......Page 90
Locations of Probe Sequence Target Regions: Discrimination of Highly Similar Targets......Page 92
In Situ Synthesis vs Deposition of Presynthesized Oligonucleotides......Page 94
Microarray Specific Effects......Page 95
Models for Hybridization in Solution......Page 99
Outlook......Page 102
References......Page 103
Introduction......Page 108
General Considerations......Page 109
Automation......Page 111
Contamination......Page 113
Purity and Quality Control......Page 114
Information Management......Page 115
Workflow......Page 117
PCR Amplification......Page 118
Purification of PCR Product......Page 120
Visualization of Purified PCR Products by Electrophoresis......Page 121
Quality Control of PCR Products......Page 124
Flag_QC......Page 125
Consolidation......Page 126
Summary......Page 128
References......Page 129
Introduction......Page 130
The Printing Process and Equipment......Page 131
Closed Capillary Pins......Page 133
Micromachined Open Capillary Pins......Page 134
Printing System Optimization......Page 135
Robotic System Issues......Page 138
Reagent-Related Failures......Page 139
Printed Spot Presence and Uniformity......Page 140
Probe Carryover......Page 141
Future Developments......Page 142
Acknowledgment......Page 143
References......Page 144
Making and Using Spotted DNA Microarrays in an Academic Core Laboratory......Page 145
Introduction......Page 146
Technologies and Services Provided by the Keck Microarray Resource......Page 147
Generic Glass Slide Microarray Printing......Page 151
Genomic Solutions OmniGrid 100 Microarrayer Print Settings......Page 152
Pin-Cleaning Procedure......Page 153
Printing Q/C Begins with Printing in Class 100 Clean Rooms for Stringent Particle Control......Page 154
First-Strand cDNA Synthesis Q/C......Page 155
Labeling and Hybridization Protocols Employed by the Keck Microarray Resource......Page 156
General Considerations Regarding RNA Samples, Labeling, and Hybridization Strategies......Page 160
Hybridization Strategies......Page 161
Total RNA and mRNA Preparation......Page 162
Target Labeling Using Aminoallyl dUTP and Monofunctional Dye Conjugation......Page 163
Target Denaturation and Hybridization......Page 164
cDNA Synthesis from Total RNA......Page 165
Post-cDNA Hybridization Wash......Page 166
Array Prehybridization......Page 167
Post-cDNA Hybridization Wash......Page 168
Genicon/Invitrogen Resonance Light Scattering Using Gold and Silver Nanoparticle Labeling with HiLight Scanner Detection......Page 169
Target Preparation and Labeling......Page 170
Target Purification after Dye Conjugation Using CyScibe GFX Purification Kit......Page 171
Yale Microarray Database......Page 172
Gene Expression Studies Using Keck Microarray Slides and/or Services......Page 175
References......Page 177
Printing Your Own Inkjet Microarrays......Page 179
Development of Inkjet Arrays......Page 180
The POSAM Arrayer......Page 181
Inside the POSAM......Page 182
Designing Oligonucleotides......Page 184
Array Reusability......Page 185
Modified Oligonucleotides......Page 187
Substrate Preparation......Page 188
Hybridization and/or Primer Extension......Page 189
Slide Preparation......Page 190
Solvent Preparation......Page 192
Oligoarray Synthesis with the POSAM......Page 193
Double-Stranded DNA by Primer Extension......Page 195
Hybridization......Page 196
Concluding Remarks......Page 197
References......Page 198
Introduction......Page 201
Experimental Section......Page 203
General......Page 204
(1S,2S)-2-(tert-Butoxycarbonylamino)-cyclopentyl Isocyanate (2)......Page 205
tert-Butyl (1S,2S)-2-[(methoxycarbonyl)methylamino)]-cyclopentyl Carbamate (4)......Page 206
N-[(2S)-tert-Butoxycarbonylaminocyclopent-(1S)-yl]-N-[thymin-1-ylacetyl]-glycine (6)......Page 207
Downloading Resin......Page 208
Cyclopentane PNA Synthesis......Page 209
Cleavage of PNA from Resin......Page 210
References......Page 211
Introduction......Page 213
RNA Type and Isolation......Page 214
Labeling and Hybridization Methods......Page 215
Choice of Samples to Compare......Page 217
Quantification and Analysis Parameters......Page 218
Sensitivity......Page 222
Reproducibility......Page 223
Statistical Analysis......Page 224
Perspectives and Conclusions......Page 228
References......Page 230
Sample Labeling: An Overview......Page 232
RNA......Page 233
RNA......Page 236
DNA......Page 238
Chemical Methods......Page 239
Enzymatic Methods......Page 241
Conclusions......Page 243
References......Page 244
Genomic DNA as a General Cohybridization Standard for Ratiometric Microarrays......Page 248
Introduction......Page 249
DNA Standards......Page 251
Recovering Information on RNA Prevalence......Page 252
Shelf Life and Quality Control for Genomic DNA Standards......Page 253
Genomic DNA Preparation......Page 254
Equipment and Reagents......Page 256
Tissue Lysis......Page 257
Genomic DNA Precipitation and Resuspension......Page 258
Column Cleanup......Page 259
Labeling Method I. Direct Incorporation of Cy-Labeled dCTP......Page 260
Denaturation and Labeling......Page 261
Labeling......Page 262
Prepare the Labeled Target for the Array......Page 263
Cy3 Standards (2 ml Final Volume Each)......Page 264
Labeling Method II. Indirect Labeling via Incorporation of Amino-allyl dUTP......Page 265
Equipment and Reagents......Page 267
Protocol for Resuspension of Dry Cy Dye in DMSO (http://pga.tigr.org/sop/M004_1a.pdf)......Page 268
Cleanup over Zymo Columns (either 5 or 25 mug Capacity)......Page 269
Labeling......Page 270
Coupling to Monofunctional Reactive Dye (http://pga.tigr.org/sop/M004_1a.pdf)......Page 271
Final Cleanup over P30 Biogel Chromatography Column......Page 272
Hybridization and Washing Methods......Page 273
Analysis of Results......Page 274
Comprehensive Reference Standard for Analysis of Multiple Microarray Experiments......Page 284
Alternate Comprehensive Standards......Page 285
Other Applications for the Genomic DNA Standard......Page 286
References......Page 287
Introduction......Page 291
Overview......Page 295
Selection and Preparation of Double-Stranded DNAs to Be Printed......Page 296
Printing and Processing of Double-Stranded DNA Microarrays......Page 297
Staining Double-Stranded DNA Microarrays......Page 298
Overview......Page 299
Protein Binding Microarray Experiment Protocol......Page 300
Microarray Data Quality Control......Page 303
Quantification of Microarray Signal Intensities and Quality Control......Page 304
Normalization of Protein-Binding Signal Intensities by DNA Signal Intensities and Identification of Significantly Bound Spots......Page 306
Identification of DNA Binding Site Motiffrom Protein Binding Microarray Data......Page 308
Conclusions......Page 309
References......Page 310
Introduction......Page 312
Microarrays for Measuring Noncoding RNA Processing and Modification......Page 313
Array Design and Construction: Spotted Arrays for Monitoring ncRNA Processing and Modification......Page 314
Sample Preparation and Hybridization......Page 315
Label Each of the Samples with Fluorescent Dyes......Page 316
Data Processing and Interpretation......Page 317
Microarrays for Monitoring Alternative Splicing......Page 319
Oligonucleotide Probe Design......Page 322
Preparation of Poly(A)+ RNA......Page 323
Preparation of Allylamine-Derivatized cDNA......Page 324
CyDye Labeling of Allylamine-Derivatized cDNA......Page 325
References......Page 326
Mapping the Distribution of Chromatin Proteins by ChIP on Chip......Page 329
Introduction......Page 330
Solutions and Materials......Page 332
Formaldehyde Cross-Linking of Chromatin from Any Drosophila Tissue......Page 333
Chromatin Immunoprecipitation Procedure......Page 334
Quality Control of ChIP Samples......Page 336
Evaluation of ChIP Specificity by Southern Blot......Page 337
Microarray Design and Production of PCR Amplicons......Page 338
Printing and Processing Microarrays Prior to Hybridization......Page 340
Procedure......Page 341
Solutions and Materials......Page 342
Procedure......Page 343
Data Acquisition......Page 344
Statistical Analysis of Data......Page 346
Graphic Comparison of ChIP on Chip Data with Genome Annotations......Page 350
Acknowledgments......Page 351
References......Page 352
Introduction......Page 355
Principle of DamID......Page 356
Correcting for Untargeted Binding of Dam......Page 357
Expression Levels of Dam and Dam Fusion Proteins......Page 358
Comparison of DamID and ChIP......Page 360
Transfection of Kc Cells......Page 361
Polymerase Chain Reaction......Page 362
Transfection of Cultured Drosophila Kc Cells......Page 363
Isolation and Digestion of Genomic DNA......Page 366
DpnII Digest......Page 367
Polymerase Chain Reaction......Page 368
Sample Labeling and Microarray Hybridizations......Page 369
Data Processing and Analysis......Page 370
References......Page 371
Whole-Genome Genotyping......Page 373
Introduction......Page 374
WGG Assay Design......Page 375
BeadChip Design......Page 378
Commercial Products......Page 379
Methods......Page 382
Sample Amplification......Page 383
Amplification Protocol......Page 384
BeadChip Hybridization......Page 385
Multilayer IHC Sandwich Staining......Page 386
Conclusions......Page 388
References......Page 389
Introduction......Page 391
Genomic DNA Purification......Page 394
Labeling......Page 395
Hybridization and Detection......Page 396
Data Interpretation......Page 397
References......Page 399
Performing Quantitative Reverse-Transcribed Polymerase Chain Reaction Experiments......Page 401
Introduction......Page 402
Choice of the Enzymatic System......Page 403
Amplification and Denaturation Curves......Page 404
Checking the Amplification Yield: Efficiency......Page 407
Relative Quantification......Page 410
Proposed Reaction Medium for SYBR Green Q-PCR......Page 412
Conclusion......Page 413
References......Page 414
Introduction......Page 416
Ethical Concerns in the Use of Tissue in Biomedical Research......Page 418
Determining What Tissue to Analyze......Page 419
Construction of a Tissue Microarray......Page 420
Obtaining a Tissue Microarray from Other Sources......Page 422
Confirmation, Validation, and Determining Experimental Approach......Page 423
Immunohistochemical Assays for Validation......Page 424
In Situ Assays......Page 426
Collection and Interpretation of Data......Page 427
Analysis of Data......Page 428
Other Applications of TMAs Relating to Microarrays......Page 429
References......Page 430
Introduction......Page 432
Preparation of Core Particles: MNase Digestion of Minichromosomes......Page 434
Immunoprecipitation of Nucleosomes......Page 436
Low‐Resolution Mapping of Nucleosome Positions Using Southern Blot Hybridization......Page 437
Mapping Immunoprecipitated Nucleosomes at Maximal Resolution by Monomer Extension......Page 440
Monomer Extension......Page 441
Analysis of the Histone Modification Map......Page 443
Concluding Remarks......Page 444
References......Page 445
