Contents
Abbreviations 00
Preface 00
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 37
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 190
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 243
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 271
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 b-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 t-test. 313
8.5 Simple nonparametric statistics: the Mann-Whitney test 315