Epigenetics Protocols (Methods in Molecular Biology, v791)

✍ Scribed by Trygve O. Tollefsbol

• Publisher: Humana Press
• Year: 2011
• Tongue: English
• Leaves: 345
• Edition: 2nd
• Category: Library

✦ Synopsis

The field of epigenetics has played a major role at the forefront not only of molecular biology, but also of medical genetics and clinical medicine.  Few disciplines have experienced growth comparable to that which we have witnessed for epigenetics in the past decade. The goal of Epigenetics Protocols, Second Edition is to highlight select techniques that have been mainstays in the field as well as to cover methods that are especially relevant to extant discoveries in epigenetics.  This volume focuses on the two broad areas of epigenetics: DNA methylation and chromatin modifications, and also covers the complex topic of computational methods for epigenetic analyses which is essential to a complete understanding of the vast body of information that is being derived with the use of these newly-developed tools.  Written in the highly successful Methods in Molecular Biology™ series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.   Authoritative and accessible, Epigenetics Protocols, Second Edition serves as an ideal guide to advanced students, basic scientists and clinical researchers as well as clinicians and biotechnology investigators who wish to continue exploring this exciting and progressive research field.

✦ Table of Contents

791_Epigenetics_[2e_MMB_2011]_9781617793158......Page 1
FrontMatter......Page 2
Epigenetics Protocols......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 10
1. Introduction......Page 14
2. DNA Methylation Gene- or Region-Specific Techniques......Page 15
3. Methods of Epigenomic Analysis......Page 17
4. Techniques to Inhibit DNA Methylation and Assess DNA Methylation Activity......Page 18
5. Chromatin Immuno-precipitation-Based Protocols......Page 19
7. In Vivo RNA–Protein Interaction Assessment......Page 20
9. Knockdown of Histone Deacetylases......Page 21
11. Conclusion......Page 22
References......Page 23
1. Introduction......Page 24
2.3. PCR Purification, Cloning, and Sequencing Analysis of Target DNA Fragment......Page 27
3.2. Bisulfite Modification......Page 28
3.5. Data Interpretation......Page 29
4. Notes......Page 30
References......Page 33
1. Introduction......Page 36
2.3. DNA Modification......Page 38
3.1.3. Sample Preparation from Paraffin-Embedded Tissues......Page 39
3.3. DNA Modification Using Sodium Bisulfite......Page 40
3.4.1. Prediction of CpG Islands......Page 41
Primer Selection for MSP......Page 42
3.5.1. Direct MSP......Page 43
4. Notes......Page 44
References......Page 45
1. Introduction......Page 46
2.2. Bisulphite Treatment and Preparation of DNA Methylation Standard Series......Page 50
2.5. Template Preparation......Page 51
3.1.1. Design of Bisulphite-Specific PCR Primers......Page 52
3.1.3. Considerations for the Region Subjected to Bisulphite Pyrosequencing......Page 53
3.2.1. Sources of Unmethylated Template......Page 54
3.2.3. Bisulphite Treatment......Page 55
3.3. Bisulphite-Specific PCR......Page 56
3.5. Template Preparation......Page 57
3.6. Bisulphite Pyrosequencing......Page 58
3.8. Data Analysis......Page 59
4. Notes......Page 62
References......Page 65
1.1. Background......Page 68
1.2. Methylation-Sensitive High-Resolution Melting......Page 69
1.3. Probe-Based Quantification of Methylated Sequences......Page 71
1.4. Sensitive Melting After Real-Time Analysis: MSP......Page 72
1.5. Deciding Which Technique to Use......Page 73
3.1. PCR Primer Design......Page 74
3.1.1. Important Points Regarding Primer Design......Page 75
3.2.2. Bisulphite Modification......Page 76
3.3. PCR Setup......Page 77
3.4.2. MethyLight and SMART-MSP......Page 78
3.5.1. Introduction......Page 79
3.5.3. Melting Analysis for SMART-MSP (and MethyLight)......Page 80
4. Notes......Page 81
References......Page 83
1. Introduction......Page 86
3.1.4. CTCFBSDB Search......Page 88
3.2.4. UCSC In Silico PCR Module......Page 89
3.3.1. Gene Name(s) for DBTSS Input......Page 90
3.3.2. NCBI Nucleotide Accession#(s) Input......Page 91
3.3.3. Fasta Sequence(s) Paste Input......Page 92
3.3.6. CpG Islands Search......Page 93
3.3.7. Methylation RFLP Analysis......Page 94
3.3.8. Bisulfite Sequencing Primer Output......Page 96
4. Notes......Page 97
References......Page 99
1. Introduction......Page 102
2.3. Reverse Phase Ion Pair High-Performance Liquid Chromatography......Page 106
3.2. SNuPE Reaction......Page 107
3.4. Multiplex Option......Page 108
4. Notes......Page 109
References......Page 112
1. Introduction......Page 114
2.3. Specific Reagent for RLGS......Page 116
3.1. Manipulation of DNA......Page 117
3.5. Identification of RLGS Spots by in silico RLGS and Spot Cloning......Page 118
3.6. Confirmation of Restriction Enzyme Sites......Page 119
3.7. Confirmation of Methylation Status......Page 120
4. Notes......Page 121
5. Conclusion......Page 122
References......Page 123
1.1. DNA Methylation......Page 126
1.2. Methods for Detection of Genome-Wide DNA Methylation......Page 127
1.4. Data Processing......Page 128
3.1. The MEDME Idea......Page 129
3.2. MEDME Overview......Page 130
3.3. Step by Step Through a MEDME Session......Page 131
3.4. Applications......Page 132
3.5. Bayesian Tool for Methylation Analysis......Page 133
4. Notes......Page 134
References......Page 135
1. Introduction......Page 138
3.2. Bead Preparation......Page 142
3.7. Analyzing the Genomic Products of MIRA......Page 143
References......Page 145
1.1. Background......Page 148
1.2. Principle of LUMA......Page 149
3.1. Assessment of DNA Quality and Integrity......Page 151
3.2. Preparation of Genomic DNA for LUMA......Page 152
3.4. Pyrosequencing ® Assay......Page 153
3.5. Data Analysis......Page 154
4. Notes......Page 155
References......Page 156
1. Introduction......Page 158
2.1. Establishment and Maintenance of a Cell Line......Page 159
2.5.1. Immunolabeling of Cells with Anti-5-Methylcytidine......Page 160
3.1. Establishment of the Fibroblast Cell Culture......Page 161
3.2. Downregulation of Dnmt1 in Somatic Cells After Extended Cell Culture......Page 162
3.4. Induction of DNA Hypomethylation Using Dnmt1-Specific siRNA......Page 163
3.4.1. Optimization of the Transfection Conditions......Page 164
3.5. Analysis of Global DNA Methylation......Page 165
3.5.2. Quantification of DNA Methylation by Flow Cytometry......Page 166
4. Notes......Page 167
References......Page 168
1. Introduction......Page 170
1.1. Radioactive Biotin Microplate DNA Methylation Assay......Page 171
1.2. Bisulfite Analysis of In Vitro Methylated DNA......Page 173
1.3. Restriction-Based Fluorescence Assay......Page 174
2.1. Radioactive Biotin Microplate Assay......Page 177
2.2. Bisulfite Analysis of In Vitro Methylated DNA......Page 178
2.3. Restriction-Based Fluorescence Assay......Page 179
3.1. Radioactive Biotin Plate Assay......Page 180
3.2. Bisulfite Analysis of In Vitro Methylated DNA......Page 181
3.3. Restriction-Based Fluorescence Assay......Page 183
References......Page 188
1. Introduction......Page 192
2.2. Reagents......Page 195
2.3. Buffers......Page 196
3.3. Preparation of Chromatin from 1,000 Cells......Page 197
3.5. Immunopreci­pitation and Washes......Page 199
3.6.1. DNA Recovery from ChIP Material: Combined DNA Elution, Cross-link Reversal, Proteinase K Digestion, Followed by DNA Purification by Phenol–Chloroform–Isoamylalcohol Extraction......Page 200
3.7.1. DNA Recovery from ChIP Samples......Page 201
4. Notes......Page 202
References......Page 205
1. Introduction......Page 208
2.3. Other Stock Solutions......Page 209
3.1. Step 1: Testing the Antibodies......Page 210
3.2. Step 2: Chromatin Preparation......Page 211
3.2.1. Cell Lysis and Purification of Nuclei......Page 212
3.2.2. Chromatin Fragmentation with MNase......Page 215
3.2.3. Incubation with the Antibody......Page 217
3.3.1. Immunoprecipitation......Page 218
3.4.1. Relative Quantification (Reference Locus Method)......Page 220
3.4.2. Absolute Quantification (% Input Recovery Method)......Page 221
4. Notes......Page 222
References......Page 224
1. Introduction......Page 226
2.1. Chromatin Immunoprecipitation......Page 228
3.1. Chromatin Immunoprecipitation......Page 229
3.2. Quantitative PCR......Page 231
4. Notes......Page 233
References......Page 236
1. Introduction......Page 238
2.2. Sequential Chromatin Immunoprecipitation......Page 242
3. Methods......Page 244
3.1. Standard (Semiquantitative) PCR and Densitometry with ImageJ Software......Page 246
4. Notes......Page 247
References......Page 250
1. Introduction......Page 252
2.1.2. Immunoprecipitation......Page 256
2.2.2. PCR Purification, Cloning, and Sequencing Analysis of ChIP DNA Fragment......Page 257
3.1.2. Immunoprecipitation of Crosslinked Chromatin DNA–Protein......Page 258
3.1.3. Purification of Immunoprecipitated Chromatin DNA and ChIP-PCR......Page 259
3.3. Data Interpretation......Page 260
4. Notes......Page 261
References......Page 263
1. Introduction......Page 266
2.1. Yeast Cell Culture and Lysis......Page 268
2.3. Immuno‑ precipitation......Page 269
3.1. Preparation of Chromatin: Yeast Cells......Page 270
3.2. Preparation of Chromatin: HEK 293T Cells......Page 271
3.3. Immuno­precipitation......Page 272
3.4. Cross-Link Reversal and RNA Purification......Page 273
4. Notes......Page 274
References......Page 277
1. Introduction......Page 278
2.4. Chromatin Immunoprecipitation Reagents......Page 281
2.6. DNA Purification......Page 282
2.9. Library Control Assay......Page 283
3.2. Preparation of Chromatin......Page 284
3.3. Determination of Chromatin Size and Concentration......Page 285
3.4. Chromatin Immunoprecipitation......Page 286
3.5. Capture of Antibody/Chromatin Complexes and Reversal of Crosslinks......Page 287
3.7. ChIP Confirmation......Page 288
3.8.2. Addition of an “A” Base to the 3' End of DNA Fragments
......Page 289
3.8.4. Size Selection of DNA Fragments......Page 290
3.8.6. Library Purification......Page 291
3.9.2. Library Enrichment Confirmation......Page 292
3.10. Library Sequencing and Data Analysis......Page 293
4. Notes......Page 294
References......Page 298
1. Introduction......Page 300
2.1. Tissue Culture......Page 301
3. Methods......Page 302
3.2. Fixation......Page 303
3.3. Sonication......Page 304
3.4. Phenol–Chloroform Extraction......Page 305
3.6. Overview of FAIRE DNA Analysis......Page 306
4. Notes......Page 307
References......Page 309
1. Introduction......Page 310
2.3. Nuclear Protein Extraction......Page 314
2.7. Chromatin Immunoprecipitation......Page 315
3.2. Nuclear Protein Extraction ( See Note 2)......Page 316
3.3.2. Odyssey Western Blot ( See Note 3)......Page 317
3.5. RT-PCR to Detect the Reexpression of Epigenetically Silenced Genes by HDAC Inhibitors in Human Breast Cancer Cells......Page 318
3.6. Chromatin Immunoprecipitation to Analyze Changes in Regulatory Chromatin Marks by HDAC Inhibitors at the Specific Gene Promoters......Page 320
4. Notes......Page 321
References......Page 323
1. Introduction......Page 326
3.1. Construction of MMSDK Tag Libraries......Page 329
3.3.2. Identifying and Trimming Reads (Tags)......Page 330
3.3.3. Construction of a Simulated MluI Reference Library......Page 332
3.3.4. Mapping Tags to the Simulated MluI Reference Library......Page 335
3.3.5. Identifying Differentially Methylated Regions......Page 336
4. Notes......Page 337
References......Page 340
Index......Page 342