RNA Methodologies (eBook)

Front Cover 1
RNA Methodologies: A Laboratory Guide for Isolation and Characterization 4
Copyright Page 5
Contents 8
Preface 22
Chapter 1 RNA and the Cellular Biochemistry Revisited 26
Why Study RNA? 26
What is RNA? 27
Assembly of Polynucleotides 30
Types of RNA 33
Transcription and the Central Dogma 35
Important Plant and Animal Models for Studying Transcription 36
Promoters and Regulatory Elements 37
Gene and Genome Organization Affect Transcription 39
RNA Polymerases and the Products of Transcription 43
Messenger RNA 47
Topology of a Typical mRNA Molecule 48
5' Cap 48
Leader Sequence 50
Coding Region 50
Trailer Sequence 51
Poly(A) Tail 51
Organellar mRNAs 53
mRNA Stability, Transport, and Turnover 54
Bicistronic mRNAs 55
Prokaryotic mRNAs 55
mRNA Sequence and Structure Affect Translation 56
Levels of Gene Regulation 57
Alternative Splicing of mRNA from a Single Genetic Locus 59
Trans-splicing: mRNA Repair 60
Overview of Small RNAs 61
miRNA Regulation of Gene Expression 62
References 63
Chapter 2 RNA Isolation Strategies 70
Rationale 70
Goals in the Purification of RNA 72
Lysis Buffer Formulations 74
Gentle Lysis Buffers 74
Protocol: Isolation of Cytoplasmic RNA by Hypotonic Lysis 76
Chaotropic Lysis Buffers 81
Isolation of RNA with Guanidinium Buffers 82
Guanidinium–Acid–Phenol Extraction Techniques 83
Protocol: Guanidinium–Acid–Phenol Extraction 84
Density Gradient Centrifugation 85
Cesium Chloride 86
Protocol: Cesium Chloride (CsCl) Gradients 86
Cesium Trifluoroacetate (CsTFA) 90
Protocol: Cesium Trifluoroacetate (CsTFA) Gradients 90
Simultaneous Isolation of RNA and DNA 93
Protocol: Simultaneous Isolation of RNA and DNA 94
The Word on Kits 96
Silica Technology 97
Isolation of Cytoplasmic RNA on a Silica Column 98
Affinity Matrices 98
Other Methods 99
Protocol: Rapid Isolation of RNA with SDS and Potassium Acetate Reagents 99
Protocol: Isolation of Prokaryotic RNA 100
Protocol: Isolation of RNA from Yeast 101
Short- and Long-Term Storage of Purified RNA 103
References 104
Chapter 3 The Truth about Tissues 106
Rationale 106
Tissue Culture or Tissue? 106
Advantages of Cell Culture 107
Advantages of Tissue Samples 108
Homogenization Methods 109
Polytron Disruption 109
Dounce Homogenization 111
BeadBeater™ Technique 112
RNA Isolation Strategies for Various Organs and Tissues 112
Fresh Tissue 114
Frozen Tissue 115
Fixed Tissue 116
Protocol: LiCl–Urea Method for RNA Isolation from Tissue 117
Protocol: RNA Isolation from Lipid-Enriched Samples 120
Purification of Polysome-Engaged mRNA 121
Protocol: Isolation of Polysomal mRNA 123
Collecting Samples in the Field 124
RNA "Clean-Up" Methods 125
Troubleshooting RNA Isolation from Tissue 126
References 127
Chapter 4 Going Green: RNA and the Molecular Biology of Plants 130
Rationale 130
RNA Isolation and the Peculiarities of Plants 130
Types of RNA Produced in Plant Cells 134
Protocol: RNA Isolation from Leaf 135
Protocol: RNA Isolation from Bark 137
Protocol: RNA Isolation from Fruit 138
Strategies for RNA Isolation from Other Plant Tissues 140
Troubleshooting RNA Isolation from Plant Tissue 141
References and Suggested Reading 142
Chapter 5 Isolation of Polyadenylated RNA 146
Rationale 146
Polyadenylation 147
The Poly(A) Caveat 148
Example 1 149
Example 2 149
Selection of Polyadenylated Molecules 151
Magnetic Bead Technology for Poly(A)[sup(+)] Purification 152
Oligo (dT)-Cellulose Column Chromatography 154
Protocol: Purification of Biophysical Quantities of Poly(A)[sup(+)] RNA 155
Rapid, Non-Column Poly(A)[sup(+)] Purification 160
Protocol: Non-Column Poly(A)[sup(+)] Purification 160
References 161
Chapter 6 Quality Control for RNA Preparations 164
Rationale 164
Quality Control Technique 1: UV Spectrophotometry and Absorption Ratios 165
Spectrophotometric Methods 165
Determination of Nucleic Acid Concentration 166
Determination of Nucleic Acid Purity 168
Non-spectrophotometric Methods 170
Quality Control Technique 2: Electrophoretic Profile of the RNA 172
Protocol 174
Quality Control Technique 3: UV Shadowing 175
Protocol 176
Quality Control Technique 4: Sample Capacity to Support RT-PCR 177
Quality Control Technique 5: Northern Analysis 178
Quality Control Technique 6: Sample Capacity to Support In Vitro Translation 178
References 179
Chapter 7 Resilient Ribonucleases 180
Rationale 180
Elimination of Ribonuclease Activity 181
Latent RNase Contamination Issues 182
Types of Ribonuclease Inhibitors 183
Specific Inhibitors 183
Vanadyl Ribonucleoside Complex (VDR VRC)
RNasin® 184
Non-specific Inhibitors 185
Preparation of Equipment and Reagents 186
Diethyl Pyrocarbonate (DEPC) 187
Alternative: Sterile Water 189
Hydrogen Peroxide 189
NaOH and SDS 190
Other Reagents Used to Control Nuclease Activity 190
Guanidine Hydrochloride 190
Guanidine Thiocyanate 190
Sodium Dodecyl Sulfate 191
N-Laurylsarcosine 191
Phenol:Chloroform: Isoamyl Alcohol 191
8-Hydroxyquinoline 192
Cesium Chloride 192
Cesium Trifluoracetate 193
Proteinase K 193
RNAlater® 194
Protocol: Synthesis of VDR 194
References 195
Chapter 8 Stringency: Conditions that Influence Nucleic Acid Structure 198
Types of Double-Stranded Molecules 198
Importance of Controlling Stringency 199
Effect of Salt on Stringency 201
Effect of pH on Stringency 201
Effect of Temperature on Stringency 202
Effect of Formamide on Stringency 202
Effect of Urea on Stringency 202
References 203
Chapter 9 Electrophoresis of RNA 204
Rationale 204
Normalization of Nucleic Acids 205
Protocol: Poly(A) Normalization 208
Sample Preparation 208
RNA Denaturing Systems for Agarose Gel Electrophoresis 210
Formaldehyde Denaturation 212
Protocol: Formaldehyde Denaturing Gels 213
Urea Denaturation 214
Protocol: Urea Denaturation 216
Glyoxal/Dimethyl Sulfoxide Denaturation 216
Protocol: Glyoxalation and Electrophoresis of RNA 217
Running RNA on Non-denaturing Gels 219
Molecular Weight Standards 219
Proper Use of Size Standards 220
Ribosomal RNA 222
Gel Staining Techniques 229
Ethidium Bromide 229
SYBR® Green 232
SYBR® Gold 232
SYBR® Safe 233
GelStar® 234
Silver Staining 234
Acridine Orange 235
Methylene Blue 235
Safety Considerations and Equipment Maintenance 237
Running Agarose Gels for the First Time: A Few Tips 238
Essential Vocabulary 238
Points to Keep in Mind 238
References 241
Chapter 10 Photodocumentation and Image Analysis 246
Rationale 246
Safety First 247
Digital Image Analysis 248
Image Formats 253
Practical Considerations 254
Digital Image Analysis for Every Budget 256
Image Analysis Workshops 257
PhosphorImagers 257
Traditional Methods of Photodocumentation 258
Sample Visualization 259
Filtration 259
Tips for Optimizing Electrophoretograms 260
Inherent Limitations of Photographic and X-ray Films 263
References and Suggested Reading 264
Chapter 11 Northern Analysis 266
Rationale 266
Choice of Filter Membrane 267
Nitrocellulose 268
Nylon 269
Polyvinylidene Difluoride 270
Handling and Filter Preparation 270
Northern Transfer Techniques 271
Capillary Transfer 271
TurboBlotter™ 272
Vacuum Blotting 273
Electroblotting 274
Alkaline Blotting 274
Protocol: RNA Transfer by Passive Capillary Diffusion 275
Protocol: TurboBlotter Downward Transfer of RNA 277
Post-Transfer Handling of Filters 279
Formaldehyde Denaturing Systems 279
Glyoxal Denaturing Systems 279
Option 1 279
Option 2 280
Immobilization Techniques 280
Baking 280
Crosslinking by UV Irradiation 281
Protocol: UV Crosslinking RNA to Nylon Filters 282
Post-Fixation Handling of Filters 282
Reverse Northern Analysis 283
References 284
Chapter 12 Nucleic Acid Probe Technology 286
Rationale 286
Probe Classification 288
Selection of Labeling System 288
Isotope Labeling 290
Minimizing Decomposition Problems 291
Non-Isotopic Labeling 292
The Ubiquitous Dyes Cy3 and Cy5 292
Popular Chemiluminescence Formats 293
Biotin 293
Digoxigenin 295
Fluorescein 296
Direct Enzyme Labeling 296
DNA Probes 297
DNA Probe Synthesis 298
Polymerase Chain Reaction 299
Random Priming 299
Nick Translation 299
5' End-Labeling 300
3' End-Labeling 300
Antisense RNA Probes 301
Characteristics of RNA Probes 303
RNA Probe Synthesis 303
In Vitro transcription 303
5' End-Labeling 304
3' End-Labeling 304
Probe Purification 304
Probe Storage 305
References 306
Chapter 13 Practical Nucleic Acid Hybridization 308
Rationale 308
Factors Influencing Hybridization Kinetics and Duplex Stability 309
Temperature 310
Ionic Strength 311
pH 312
Probe Length 312
Probe Concentration 312
G+C Content 313
Mismatching 313
Probe Complexity 314
Viscosity 314
Formamide 315
Urea 315
Hybridization Temperature 316
T[sub(m)] for Long Probes 316
T[sub(m)] for Oligonucleotide Probes 317
Hybridization and the Northern Analysis 317
Prehybridization: Filter Preparation 318
Protocol: Prehybridization for Long Probes 318
Probe Denaturation 319
Hybridization 320
Posthybridization Stringency Washes 320
Probe Removal and Rehybridization 321
Protocol: Generic Method for Probe Removal 322
References 323
Chapter 14 Principles of Detection 326
Rationale 326
Autoradiography 327
Handling of Filter Membranes 329
X-ray Film 330
Safe light 331
Exposure Time 331
Intensifying Screens 332
Fluorography 333
Preflashing Film 334
Type of Cassette 334
Development and Fixation 335
Protocol: Autoradiography 335
Non-isotopic Procedures 337
Biotin 338
Digoxigenin 338
Fluorescence Imaging 339
Direct Enzyme Labeling 339
Detection by Chemiluminescence 339
Substract for Chemiluminescence 340
Chromogenic Detection Procedures 342
Digital Imaging Systems 343
References 345
Chapter 15 Quantification of Specific mRNAs by Nuclease Protection 346
Rationale 346
Basic Approach 347
Probe Selection 352
Optimization Suggestions 354
Potential Difficulties 355
Protocol: Transcript Quantification by S1 Analysis 357
Protocol: Transcript Quantification by RNase Protection 360
Troubleshooting 362
References 363
Chapter 16 Analysis of Nuclear RNA 366
Rationale 366
Transcription Rate Assays 368
Relationship to the Study of Steady-State RNA 371
Nuclear Run-off vs. Nuclear Run-on Assay 372
Protocol: Nuclear Run-off Assay 373
Harvesting of Cells and Preparation of Nuclei 374
Alternative Protocol for Preparation of Fragile Nuclei 374
Alternative Protocol for Preparation of Nuclei from Whole Tissue 375
Labeling and Recovery of Transcripts 376
Preparation of Target DNA 378
Preparation of RNA for Hybridization 379
Posthybridization Washes and Detection 380
Protocol: Alternative Procedure for Nuclear Run-off Assay 381
Protocol: Nuclease Protection–Pulse Label Transcription Assay 382
Distinguishing Among the Activities of RNA Polymerases 384
Extraction of Nuclear RNA for Steady-State Analysis 385
Protocol: Direct Isolation of Nuclear RNA 386
Protocol: Preparation of Nuclear RNA from Cells Enriched in Ribonuclease 387
Troubleshoot Nuclear RNA Analysis 388
References 389
Chapter 17 cDNA Synthesis 392
Rationale 392
cDNA Synthesis – An Overview 393
First-Strand Considerations 394
Reverse Transcriptase Options 397
Second-Strand Considerations 400
Classical Methods 401
PCR-Based Methods 402
Protocol: First-Strand cDNA Synthesis 402
Assessing cDNA Synthesis Efficiency 404
Cloning cDNA 404
Ligation Considerations 406
Enzymes Used for Ligation 408
Applications 408
References 409
Chapter 18 RT-PCR: A Science and an Art Form 410
Rationale 410
PCR – An Overview 411
RT-PCR – General Approach 416
PCR Carryover Prevention 420
Laboratory Design 420
Procedural Methods 421
Aerosol-Resistant Tips 422
Uracil-N-Glycosylase 423
Primer Design 423
Basic Rules 427
T[sub(m)] Considerations 431
Estimating T[sub(m)] 431
Precision T[sub(m)] Calculations 432
On-line Resources 432
Multiplex Primer Design 432
Optimization Procedures 433
Thermostable Polymerases 436
Positive Controls 438
Negative Controls 438
Hot Start PCR 438
Locked Nucleic Acids 440
Touchdown PCR 441
Internal Controls 441
The Word on Transcription Controls 444
Analysis of PCR Products 445
RT-PCR Quality Control Points 446
Non-PCR Methods for Confirming PCR-derived Data 448
Related Techniques 449
5' RACE PCR 449
5' RLM-RACE 451
3' RACE PCR 451
Nested PCR 453
Long-Range PCR 454
Single Cell PCR 456
Splinkerette PCR 458
The Hunt for Alternative Transcription Start Sites 459
Protocol: First-Strand cDNA Synthesis 460
Protocol: PCR Amplification of cDNA 461
Cloning PCR Products 462
Protocol: A-Tailing of Blunt-End PCR Products 463
Protocol: TA Cloning Ligation Reaction 463
TOPO® Cloning 465
Other Amplification Procedures 466
Linear RNA Amplification (Eberwine Process) 466
Strand Displacement Amplification 467
Nucleic Acid Sequence Based Amplification (NASBA) 468
Ligase Chain Reaction 468
References 468
Chapter 19 Quantitative PCR Techniques 474
Rationale 474
Sensitivity Index 475
Quantitative Approaches 476
Real-Time PCR 478
Real-Time PCR Platforms 481
SYBR Green Assay 482
TaqMan® Assay 483
Molecular Beacons 483
LUX™ Primers 485
Scorpions 486
Melting Curve Analysis 487
Internal Controls 488
Exogenous Controls 488
Control Reaction Formats 492
Negative Control Considerations 496
Competitive PCR: Key Considerations 497
Competitive PCR: Major Steps Involved 502
Alternative Approach: Non-Real-Time Competitive PCR 504
Protocol: Competitive PCR 505
Synthesis of Non-homologous Competitor 505
Synthesis of First-Strand cDNA 506
Competitive PCR (Primary Amplification) 507
Competitive PCR (Secondary Amplification) 509
Image Analysis Considerations 510
Troubleshooting Quantitative PCR Techniques 511
References 513
Chapter 20 Functional Genomics and Transcript Profiling 516
Rationale 516
Functional Genomics Defined 516
Importance of Functional Genomics Approaches 517
Commonly Used Functional Genomics Approaches 518
Functional Genomics and Classical Molecular Biology 521
Chapter 21 High-Throughput Analysis of Gene Expression 524
Rationale 524
What is a Microarray? 525
What is a Heat Map? 527
What Microarrays Can Do 529
What Microarrays Cannot Do 529
Major Steps in Microarray Analysis 532
Reference RNA 534
What is a Macroarray? 535
Applications 537
References 537
Chapter 22 Non-Array Methods for Global Analysis of Gene Expression 540
Rationale 540
Essential Issues 541
Subtractive Method 542
Suppression Subtractive Hybridization (SSH) 542
Troubleshooting 549
Non-Subtractive Methods 551
mRNA Differential Display 551
Protocols 554
References 561
Chapter 23 RNAi: Take a RISC – Role the Dicer 564
Rationale 564
Essential RNAi Nomenclature 566
RNA Interference – How it Works 567
siRNA Approach 570
shRNA Approach 571
siRNA Delivery Methods into Mammalian Cells 573
miRNA 574
Effective Design of siRNAs 575
RNAi and Alternative Transcript Splicing 577
In Vitro and In Vivo Issues 578
RNAi Validation 580
RT-PCR Approaches 581
Northern Analysis 581
Western Analysis 581
Applications 582
References 582
Chapter 24 Genomes, Transcriptomes, Proteomes, and Bioinformatics 586
Rationale 586
Essential Nomenclature 588
Genomes and Genomics 589
Transcriptomes and Transcriptomics 590
Proteomes and Proteomics 591
Bioinformatics 596
Search for Genes – Have a BLAST! 596
References 598
Chapter 25 An RNA Paradigm 600
A Typical Experiment? 602
Sensitivity Issues 605
What to Do Next 605
Where to Turn for Help 607
Epilogue: A Few Pearls of Wisdom 610
Appendix A: Maintaining Complete and Accurate Records 616
Appendix B: Converting Mass to Moles 618
Appendix C: Useful Stock Solutions for the Molecular Biologist 622
Appendix D: Phenol Preparation 628
Appendix E: Disposal of Ethidium Bromide and SYBR Green Solutions 632
Appendix F: DNase I Removal of DNA from an RNA Sample 636
Appendix G: RNase Incubation to Remove RNA from a DNA Sample 638
Appendix H: Deionization of Formamide, Formaldehyde, and Glyoxal 640
Appendix I: Silanizing Centrifuge Tubes and Glassware 642
Appendix J: Trypsinization Protocol for Anchorage-Dependent Cells 644
Appendix K: Isolation of High-Molecular-Weight DNA by Salting-Out 646
Appendix L: Electrophoresis: Principles, Parameters, and Safety 650
Appendix M: Polyacrylamide Gel Electrophoresis 662
Appendix N: Dot Blot Analysis 666
Appendix O: Centrifugation as a Mainstream Tool for the Molecular Biologist 674
Appendix P: Selected Suppliers of Equipment, Reagents, and Services 680
Appendix Q: Useful SI Units 688
Appendix R: Common Abbreviations 690
Appendix S: Trademark Citations 692
Glossary 696
A 696
B 697
C 698
D 701
E 702
F 703
G 703
H 704
I 705
J 705
K 705
L 705
M 706
N 707
O 708
P 708
Q 711
R 711
S 712
T 714
U 716
V 716
W 717
X 717
Z 717
Index 718
A 718
B 719
C 719
D 721
E 723
F 725
G 726
H 727
I 728
J 728
K 728
L 728
M 729
N 730
O 733
P 733
Q 736
R 737
S 739
T 741
U 742
V 742
W 742
X 742
Y 742