| Abbreviations | p. ix |
| Preface | p. xi |
| Gene expression and its control | p. 1 |
| Introduction | p. 1 |
| An overview of the mechanics of transcription | p. 5 |
| Post-transcriptional modification, processing and nuclear export of coding RNA | p. 14 |
| An overview of the mechanism of translation | p. 17 |
| Control of transcription in prokaryotes | p. 23 |
| Control of transcription in eukaryotes | p. 29 |
| Post-transcriptional control of gene expression | p. 35 |
| Further reading | p. 39 |
| References | p. 39 |
| Isolation and analysis of RNA | p. 41 |
| Introduction | p. 41 |
| The properties of different types of RNA | p. 41 |
| Purification of RNA: an introduction | p. 44 |
| Stabilizing the RNA complement prior to harvesting cells | p. 48 |
| Harvesting and lysing or disrupting cells | p. 51 |
| Methods for the isolation and purification of RNA | p. 56 |
| Re-solubilization and storage of RNA | p. 71 |
| Quantification of RNA concentration using a spectrophotometer | p. 72 |
| Sources of contamination in RNA preparations and how to spot them | p. 73 |
| Separation of RNA samples using electrophoresis | p. 75 |
| Analysis of RNA molecules using the Bioanalyser | p. 87 |
| Further reading | p. 89 |
| References | p. 89 |
| Isolation of RNA from animal cells using the acid phenol method | p. 91 |
| Isolation of RNA from bacterial and yeast cells using guanidine isothiocyanate and lithium chloride precipitation | p. 93 |
| Isolation of RNA from plant and filamentous fungal cells by using guanidine hydrochloride | p. 95 |
| Rapid isolation of RNA from animal tissues and cells using guanidine isothiocyanate, lithium chloride and cesium trifluoroacetate isopycnic density centrifugation | p. 96 |
| Separate isolation of cytoplasmic and nuclear RNA from tissue culture cells | p. 98 |
| Purification of RNA using silica beads | p. 99 |
| Hybridization-based methods for measuring transcript levels | p. 101 |
| The basics of nucleic acid hybridization | p. 101 |
| Blotting | p. 104 |
| Using hybridization to quantify RNA molecules | p. 109 |
| The northern blot | p. 109 |
| Making DNA probes | p. 111 |
| Northern hybridization reactions | p. 116 |
| Visualization of target:probe interactions | p. 120 |
| Limitations and design of northern blot hybridization experiments and interpretation of the results | p. 124 |
| Northern hybridization meets Elisa | p. 129 |
| Array-based hybridization methods | p. 131 |
| Types of array | p. 132 |
| Labeled targets for array hybridization | p. 134 |
| Probes for array-based hybridization | p. 143 |
| Experimental and data analysis approaches for use with array hybridization | p. 149 |
| Limitations and design of micro-array transcriptomics experiments | p. 151 |
| Nuclear run-off assays | p. 157 |
| References | p. 159 |
| Production of a DNA probe | p. 160 |
| 5[prime] end-labeling of oligonucleotides | p. 161 |
| Synthesis of first-strand cDNA | p. 162 |
| Synthesis of second-strand cDNA | p. 163 |
| Ligation of linker sequences to blunt-ended cDNA | p. 164 |
| RNA production in vitro for cDNA amplification | p. 165 |
| Nuclear run-off assay | p. 166 |
| PCR-based methods for measuring transcript levels | p. 167 |
| Introduction | p. 167 |
| The basics of PCR | p. 167 |
| Methodological aspects of PCR experiments | p. 175 |
| Analysis of PCR products using agarose gel electrophoresis | p. 184 |
| Problems with, and optimization of, PCR | p. 185 |
| Quantitative PCR | p. 189 |
| Real-time PCR: the basics | p. 191 |
| Reverse transcription-PCR measurement of RNA levels (qRT-PCR) | p. 200 |
| qRT-PCR methodologies | p. 200 |
| SIP-PCR and the Virtual northern blot | p. 205 |
| In situ qRT-PCR | p. 208 |
| Further reading | p. 209 |
| References | p. 210 |
| PCR amplification of a specific cDNA sequence | p. 212 |
| Single-tube RT-PCR | p. 213 |
| PolyA plus SIP-PCR | p. 214 |
| Virtual northern blot | p. 215 |
| Differential display, subtractive hybridization, amplification suppression and SAGE techniques for measuring gene expression | p. 217 |
| Introduction | p. 217 |
| Determining differences between genomic complements | p. 218 |
| Differential display techniques for measuring gene expression | p. 221 |
| Subtractive hybridization techniques for measuring gene expression | p. 226 |
| Amplification suppression techniques for measuring gene expression | p. 231 |
| Serial analysis of gene expression | p. 238 |
| Further reading | p. 242 |
| References | p. 242 |
| Measuring gene expression using reporter gene assays | p. 245 |
| Introduction | p. 245 |
| A guide to measuring enzyme activity | p. 247 |
| Western blotting: a beginner's guide | p. 253 |
| Promoter-probe reporter enzymes and assay of their activity | p. 265 |
| Using promoter-probe reporter vectors | p. 270 |
| End-product gene expression reporter assays | p. 272 |
| Making reporter gene fusions for end-product gene expression studies | p. 277 |
| References | p. 277 |
| The Bradford protein assay | p. 279 |
| Simplified assay of [Beta]-galactosidase activity | p. 280 |
| Analysis of the proteome | p. 281 |
| Direct methods for calculating the relative amounts of a known protein in different cell extracts | p. 281 |
| Separating a test protein from the rest of the proteome using two-dimensional gel electrophoresis | p. 287 |
| Determining changes in the proteome | p. 296 |
| Identifying proteins | p. 303 |
| Further reading | p. 308 |
| References | p. 308 |
| Elisa analysis | p. 309 |
| Cyanogen bromide cleavage of insoluble proteins | p. 310 |
| Statistical analysis of gene expression data | p. 311 |
| Statistical analysis: what is the point? | p. 311 |
| Standard deviations | p. 312 |
| The normal distribution | p. 313 |
| Simple parametric statistics: the t-test | p. 313 |
| Simple nonparametric statistics: the Mann-Whitney test | p. 315 |
| Index | p. 319 |
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