RNA methodologies : a laboratory guide for isolation and characterization

Contents Preface xxi 1 RNA and the Cellular Biochemistry Revisited 1 Why Study RNA? 1 What is RNA? 2 Assembly of Polynucleotides 5 Types of RNA 8 Transcription and the Central Dogma 10 Important Plant and Animal Models for Studying Transcription 11 Promoters and Regulatory Elements 12 Gene and Genome Organization Affect Transcription 14 RNA Polymerases and the Products of Transcription 18 Messenger RNA 22 Topology of a Typical mRNA Molecule 23 5  Cap 23 Leader Sequence 25 Coding Region 25 Trailer Sequence 26 Poly(A) Tail 26 Organellar mRNAs 28 mRNA Stability, Transport, and Turnover 29 Bicistronic mRNAs 30 Prokaryotic mRNAs 30 mRNA Sequence and Structure Affect Translation 31 Levels of Gene Regulation 32 Alternative Splicing of mRNA from a Single Genetic Locus 34 Trans -splicing: mRNA Repair 35 Overview of Small RNAs 36 miRNA Regulation of Gene Expression 37 References 38 2 RNA Isolation Strategies 45 Rationale 45 Goals in the Purification of RNA 47 Lysis Buffer Formulations 49 Gentle Lysis Buffers 49 Troubleshooting RNA Isolation from Tissue 101 References 102 4 Going Green: RNA and the Molecular Biology of Plants 105 Rationale 105 RNA Isolation and the Peculiarities of Plants 105 Types of RNA Produced in Plant Cells 109 Protocol : RNA Isolation from Leaf 110 Protocol : RNA Isolation from Bark 112 Protocol : RNA Isolation from Fruit 113 Strategies for RNA Isolation from Other Plant Tissues 115 Troubleshooting RNA Isolation from Plant Tissue 116 References and Suggested Reading 117 5 Isolation of Polyadenylated RNA 121 Rationale 121 Polyadenylation 122 The Poly(A) Caveat 123 Example 1 124 Example 2 124 Selection of Polyadenylated Molecules 126 Magnetic Bead Technology for Poly(A)  Purification 127 Oligo (dT)-Cellulose Column Chromatography 129 Protocol: Purification of Biophysical Quantities of Poly(A)  RNA 130 Rapid, Non-Column Poly(A)  Purification 135 Protocol: Non-Column Poly(A)  Purification 135 References 136 6 Quality Control for RNA Preparations 139 Rationale 139 Quality Control Technique 1: UV Spectrophotometry and Absorption Ratios 140 Spectrophotometric Methods 140 Determination of Nucleic Acid Concentration 141 Determination of Nucleic Acid Purity 143 Non-spectrophotometric Methods 145 Quality Control Technique 2: Electrophoretic Profile of the RNA 147 Protocol 149 Quality Control Technique 3: UV Shadowing 150 Protocol 151 Quality Control Technique 4: Sample Capacity to Support RT-PCR 152 Quality Control Technique 5: Northern Analysis 153 Quality Control Technique 6: Sample Capacity to Support In Vitro Translation 153 References 154 Contents xi Protocol: Formaldehyde Denaturing Gels 188 Urea Denaturation 189 Protocol: Urea Denaturation 191 Glyoxal/Dimethyl Sulfoxide Denaturation 191 Protocol: Glyoxalation and Electrophoresis of RNA 192 Running RNA on Non-denaturing Gels 194 Molecular Weight Standards 194 Proper Use of Size Standards 195 Ribosomal RNA 197 Gel Staining Techniques 204 Ethidium Bromide 204 SYBR® Green 207 SYBR® Gold 207 SYBR® Safe 208 GelStar® 209 Silver Staining 209 Acridine Orange 210 Methylene Blue 210 Safety Considerations and Equipment Maintenance 212 Running Agarose Gels for the First Time: A Few Tips 213 Essential Vocabulary 213 Points to Keep in Mind 213 References 216 10 Photodocumentation and Image Analysis 221 Rationale 221 Safety First 222 Digital Image Analysis 223 Image Formats 228 Practical Considerations 229 Digital Image Analysis for Every Budget 231 Image Analysis Workshops 232 PhosphorImagers 232 Traditional Methods of Photodocumentation 233 Sample Visualization 234 Filtration 234 Tips for Optimizing Electrophoretograms 235 Inherent Limitations of Photographic and X-ray Films 238 References and Suggested Reading 239 11 Northern Analysis 241 Rationale 241 Choice of Filter Membrane 242 Nitrocellulose 243 Nylon 244 Polyvinylidene Difluoride 245