Abbreviations .................................................. ix
Preface ........................................................ xi
1. Gene expression and its control ............................. 1
1.1. Introduction .......................................... 1
1.2. An overview of the mechanics of transcription ......... 5
1.3. Post-transcriptional modification, processing and
nuclear export of coding RNA ......................... 14
1.4. An overview of the mechanism of translation .......... 17
1.5. Control of transcription in prokaryotes .............. 23
1.6. Control of transcription in eukaryotes ............... 29
1.7. Post-transcriptional control of gene expression ...... 35
Further reading ............................................ 39
References ................................................. 39
2. Isolation and analysis of RNA .............................. 41
2.1. Introduction ......................................... 41
2.2. The properties of different types of RNA ............. 41
2.3. Purification of RNA: an introduction ................. 44
2.4. Stabilizing the RNA complement prior to harvesting
cells ................................................ 48
2.5. Harvesting and lysing or disrupting cells ............ 51
2.6. Methods for the isolation and purification of RNA .... 56
2.7. Re-solubilization and storage of RNA ................. 71
2.8. Quantification of RNA concentration using a
spectrophotometer .................................... 72
2.9. Sources of contamination in RNA preparations and
how to spot them ..................................... 73
2.10. Separation of RNA samples using electrophoresis ...... 75
2.11. Analysis of RNA molecules using the Bioanalyser ...... 87
Further reading ............................................ 89
References ................................................. 89
Protocol 2.1. Isolation of RNA from animal cells using
the acid phenol method ....................... 91
Protocol 2.2. Isolation of RNA from bacterial and yeast
cells using guanidine isothiocyanate and
lithium chloride precipitation ............... 93
Protocol 2.3. Isolation of RNA from plant and filamentous
fungal cells by using guanidine
hydrochloride ................................ 95
Protocol 2.4. Rapid isolation of RNA from animal tissues
and cells using guanidine isothiocyanate,
lithium chloride and cesium
trifluoroacetate isopycnic density
centrifugation ............................... 96
Protocol 2.5. Separate isolation of cytoplasmic and
nuclear RNA from tissue culture cells ........ 98
Protocol 2.6. Purification of RNA using silica beads ....... 99
3. Hybridization-based methods for measuring transcript
levels .................................................... 101
3.1. The basics of nucleic acid hybridization ............ 101
3.2. Blotting ............................................ 104
3.3. Using hybridization to quantify RNA molecules ....... 109
3.4. The northern blot ................................... 109
3.5. Making DNA probes ................................... 111
3.6. Northern hybridization reactions .................... 116
3.7. Visualization of target:probe interactions .......... 120
3.8. Limitations and design of northern blot
hybridization experiments and interpretation of
the results ......................................... 124
3.9. Northern hybridization meets ELISA .................. 129
3.10. Array-based hybridization methods ................... 131
3.11. Types of array ...................................... 132
3.12. Labeled targets for array hybridization ............. 134
3.13. Probes for array-based hybridization ................ 143
3.14. Experimental and data analysis approaches for use
with array hybridization ............................ 149
3.15. Limitations and design of micro-array
transcriptomics experiments ......................... 151
3.16. Nuclear run-off assays .............................. 157
References ................................................ 159
Protocol 3.1. Production of a DNA probe ................... 160
Protocol 3.2. 5' end-labeling of oligonucleotides ......... 161
Protocol 3.3. Synthesis of first-strand cDNA .............. 162
Protocol 3.4. Synthesis of second-strand cDNA ............. 163
Protocol 3.5. Ligation of linker sequences to blunt-
ended cDNA .................................. 164
Protocol 3.6. RNA production in vitro for cDNA
amplification ............................... 165
Protocol 3.7. Nuclear run-off assay ....................... 166
4. PCR-based methods for measuring transcript levels ......... 167
4.1. Introduction ........................................ 167
4.2. The basics of PCR ................................... 167
4.3. Methodological aspects of PCR experiments ........... 175
4.4. Analysis of PCR products using agarose gel
electrophoresis ..................................... 184
4.5. Problems with, and optimization of, PCR ............. 185
4.6. Quantitative PCR .................................... 189
4.7. Real-time PCR: the basics ........................... 191
4.8. Reverse transcription-PCR measurement of RNA
levels (qRT-PCR) .................................... 200
4.9. qRT-PCR methodologies ............................... 200
4.10. SIP-PCR and the virtual northern blot ............... 205
4.11. In situ qRT-PCR ..................................... 208
Further reading ........................................... 209
References ................................................ 210
Protocol 4.1. PCR amplification of a specific cDNA
sequence .................................... 212
Protocol 4.2. Single-tube RT-PCR .......................... 213
Protocol 4.3. PolyA plus SIP-PCR .......................... 214
Protocol 4.4. Virtual northern blot ....................... 215
5. Differential display, subtractive hybridization,
amplification suppression and SAGE techniques for
measuring gene expression ................................. 217
5.1. Introduction ........................................ 217
5.2. Determining differences between genomic
complements ......................................... 218
5.3. Differential display techniques for measuring gene
expression .......................................... 221
5.4. Subtractive hybridization techniques for measuring
gene expression ..................................... 226
5.5. Amplification suppression techniques for measuring
gene expression ..................................... 231
5.6. Serial analysis of gene expression .................. 238
Further reading ........................................... 242
References ................................................ 242
6. Measuring gene expression using reporter gene assays ...... 245
6.1. Introduction ........................................ 245
6.2. A guide to measuring enzyme activity ................ 247
6.3. Western blotting: a beginner's guide ................ 253
6.4. Promoter-probe reporter enzymes and assay of their
activity ............................................ 265
6.5. Using promoter-probe reporter vectors ............... 270
6.6. End-product gene expression reporter assays ......... 272
6.7. Making reporter gene fusions for end-product gene
expression studies .................................. 277
References ................................................ 277
Protocol 6.1. The Bradford protein assay .................. 279
Protocol 6.2. Simplified assay of p-galactosidase
activity .................................... 280
7. Analysis of the proteome .................................. 281
7.1. Direct methods for calculating the relative
amounts of a known protein in different cell
extracts ............................................ 281
7.2. Separating a test protein from the rest of the
proteome using two-dimensional gel
electrophoresis ..................................... 287
7.3. Determining changes in the proteome ................. 296
7.4. Identifying proteins ................................. 303
Further reading ........................................... 308
References ................................................ 308
Protocol 7.1. ELISA analysis .............................. 309
Protocol 7.2. Cyanogen bromide cleavage of insoluble
proteins .................................... 310
8. Statistical analysis of gene expression data .............. 311
8.1. Statistical analysis: what is the point? ............ 311
8.2. Standard deviations ................................. 312
8.3. The normal distribution ............................. 313
8.4. Simple parametric statistics: the r-test ............ 313
8.5. Simple nonparametric statistics: the Mann-Whitney
test ................................................ 315
Index ......................................................... 319
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