RNA methodologies: a laboratory guide for isolation and characterization
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
San Diego [u.a.]
Acad. Press
1998
|
| Ausgabe: | 2. ed. |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XX, 533 S. Ill., graph. Darst. |
| ISBN: | 0122496957 |
Internformat
MARC
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| 245 | 1 | 0 | |a RNA methodologies |b a laboratory guide for isolation and characterization |c Robert E. Farrell |
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Datensatz im Suchindex
| _version_ | 1804126886169673728 |
|---|---|
| adam_text | Contents
Preface xix
CHAPTER 1
RNA and the Cellular Biochemistry Revisited
Why Study RNA? 2
What Is RNA? 3
Assembly of Polynucleotides 7
Stringency: Conditions That Influence Nucleic Acid Structure 9
Effect of Salt on Stringency 10
Effect of pH on Stringency 11
Effect of Temperature on Stringency 11
Effect of Formamide on Stringency 11
Types of Double Stranded Molecules 12
References 12
CHAPTER 2
Transcription and the Organization of Eukaryotic
Gene Sequences
Transcription and the Central Dogma 14
Regulatory Elements 14
DNA Template 15
Gene Organization 16
RNA Polymerases and the Products of Transcription 18
References 21
viii Contents
CHAPTER 3
Messenger RNA
Topology of a Typical mRNA Molecule 24
5 Cap 25
Leader Sequence 26
Coding Region 27
Trailer Sequence 27
Poly(A) Tail 27
Organellar mRNAs 29
Stability in the Cytoplasm 30
Levels of Regulation 30
References 33
CHAPTER 4
Resilient Ribonucleases
Rationale 37
Elimination of Ribonuclease Activity 37
Types of Ribonuclease Inhibitors 38
Specific Inhibitors 39
Nonspecific Inhibitors 41
Preparation of Equipment and Reagents 41
Diethyl Pyrocarbonate 43
Alternative: Sterile Water for Irrigation 45
Hydrogen Peroxide 45
NaOH and SDS 46
Other Reagents Used to Control Nuclease Activity 46
Guanidine Hydrochloride 46
Guanidine Thiocyanate 46
Sodium Dodecyl Sulfate 47
A Laurylsarcosine 47
Phenol:Chloroform:Isoamyl Alcohol 47
8 Hydroxyquinoline 48
Cesium Chloride 49
Cesium Trifluoroacetate 49
Proteinase K 49
Protocol: Synthesis of VDR 50
References 52
Contents ix
CHAPTER 5
RNA Isolation Strategies
Rationale 55
Goals in the Purification of RNA 56
Chaotropic Lysis Buffers 59
Gentle Lysis Buffers 60
Protocol: Isolation of Cytoplasmic RNA by Hypotonic Lysis
withNP 40 61
Advance Preparation of Extraction Buffer 62
RNA Isolation 64
Isolation of RNA with Guanidinium Buffers 67
Isopycnic Centrifugation 69
Guanidinium Acid Phenol Techniques 80
Protocol: Rapid Isolation of RNA with SDS and Potassium Acetate
Reagents 85
Protocol: Isolation of Prokaryotic RNA 86
Protocol: Simultaneous Isolation of RNA and DNA 88
Recovery of RNA 90
Recovery of DNA 91
Protocol: Isolation of RNA from Yeast 92
Determination of Nucleic Acid Concentration and Purity 94
Spectrophotometric Methods 94
Nonspectrophotometric Methods 98
Short and Long Term Storage of Purified RNA 100
References 101
CHAPTER 6
Isolation of Polyadenylated RNA
Rationale 105
The Poly(A) Caveat 106
Polyadenylation 108
Selection of Polyadenylated Molecules 109
Oligo(dT) Cellulose Column Chromatography 110
Protocol: Purification of Biophysical Quantities of Poly(A)+ RNA 112
Protocol: Rapid, Noncolumn Poly(A)+ Purification 117
Magnetic Bead Technology for Poly(A)+ Purification 120
Protocol: Purification of Poly(A)+ RNA with Magnetic Beads 123
References 125
x Contents
CHAPTER 7
Quality Control for RNA Preparations
Rationale 129
Quality Control Technique 1 129
Protocol 131
Quality Control Technique 2 133
Quality Control Technique 3 133
Quality Control Technique 4 133
Quality Control Technique 5 134
CHAPTER 8
Dot Blot Analysis
Rationale 136
Advantages and Disadvantages 136
Appropriate Positive and Negative Controls 139
Protocol: RNA Dot Blots 140
Protocol: DNA Dot Blots 141
Limitations of the Data 143
References 143
CHAPTER 9
Electrophoresis of RNA
Rationale 145
RNA Denaturing Systems for Agarose Gel Electrophoresis 147
Normalization of Nucleic Acids 148
Protocol 150
Formaldehyde 153
Protocol 153
Glyoxal/Dimethyl Sulfoxide 156
Protocol 157
Molecular Weight Standards 160
Proper Use of Standards 162
Gel Staining Techniques 168
Ethidium Bromide 168
SYBR Green 170
Silver Staining 170
Acridine Orange 172
Methylene Blue 173
Contents xi
Running Agarose Gels for the First Time: A Few Tips 174
Essential Vocabulary 174
Points to Keep in Mind 174
References 177
CHAPTER 10
Photodocumentation and Image Analysis
Rationale 181
Photodocumentation 181
Sample Visualization 183
Filtration 184
Safety First 185
Tips for Optimizing Electrophoretograms 186
Inherent Limitations of Photographic Film 189
Digital Image Analysis 190
Image Formats 195
Practical Considerations 195
References 199
CHAPTER 11
The Northern Analysis
Rationale 201
Choice of Filter Membrane 201
Nitrocellulose 202
Nylon 203
Polyvinylidene Difluoride 203
Handling and Filter Preparation 204
Northern Transfer Techniques 204
Capillary Transfer 205
TurboBlotter 206
Vacuum Blotting 207
Positive Pressure 208
Electroblotting 208
Alkaline Blotting 209
Protocol: RNA Transfer by Passive Capillary Diffusion 209
Protocol: TurboBlotter Downward Transfer of RNA 212
Posttransfer Handling of Filters 215
Formaldehyde Denaturing Systems 215
Glyoxal Denaturing Systems 216
Immobilization Techniques 216
Baking 216
xii Contents
Crosslinking by UV Irradiation 217
Postf ixation Handling of Filters 219
References 219
CHAPTER 12
Nucleic Acid Probe Technology
Rationale 222
Probe Classification 223
Selection of Labeling System 223
Isotopic Labeling 224
Nonisotopic Labeling 229
DNA Probes 232
DNA Probe Synthesis 233
RNA Probes 237
Characteristics of RNA Probes 239
RNA Probe Synthesis 240
Probe Purification 241
Probe Storage 242
References 243
CHAPTER 13
Practical Nucleic Acid Hybridization
Rationale 246
Factors Influencing Hybridization Kinetics and Specificity 247
Temperature 247
Ionic Strength 248
pH 249
Probe Length 249
Probe Concentration 249
Guanosine and Cytosine Content 250
Mismatching 251
Probe Complexity 251
Viscosity 251
Formamide 252
Hybridization Temperature 252
Tm for Long Probes 253
Tm for Oligonucleotide Probes 253
Hybridization and the Northern Analysis 254
Prehybridization: Filter Preparation 254
Protocol: Prehybridization (Long Probes) 255
Probe Denaturation 257
Contents xiii
Hybridization 257
Posthybridization Stringency Washes 257
References 258
CHAPTER 14
Principles of Detection
Rationale 261
Autoradiography 262
Handling of Filter Membranes 264
X Ray Film 265
Exposure Time 266
Intensifying Screens 266
Fluorography 268
Preflashing Film 268
Type of Cassette 268
Development and Fixation 269
Autoradiography: Suggested Protocol 269
Nonisotopic Procedures 272
Biotin 273
Digoxigenin 275
Fluorescein 275
Direct Enzyme Labeling 276
Detection by Chemiluminescence 276
Chromogenic Detection Procedures 279
References 280
CHAPTER 15
RT PCR
Rationale 283
cDNA Synthesis—An Overview 283
First Strand Considerations 285
Second Strand Considerations 289
Ligation Considerations 291
PCR—An Overview 293
RT PCR—General Approach 297
Primer Design 300
Tm Considerations 304
Optimization Procedures 305
Analysis of PCR Products 311
RT PCR Quality Control Points 312
Related Techniques 313
XIV Contents
5 RACEPCR 313
3 RACEPCR 314
Nested PCR 314
Protocol: First Strand cDNA Synthesis 315
Protocol: PCR Amplification of cDNA 316
Cloning PCR Products 317
Protocol: Rapid TA Cloning of PCR Products 318
TopoCloning 320
References 320
CHAPTER 16
Messenger RNA Differential Display
Rationale 324
General Approach 326
Product Variety: What to Expect 337
Protocol: mRNA Differential Display 340
Protocol: Identification and Selection of Differentially Expressed
Sequences 345
Recovery of Differentially Expressed Sequences by Affinity Capture 346
Cloning PCR Products 348
Confirmation of Differential Expression 348
Subsequent Characterization 349
Applications of Differential Display 350
Trouble Shooting mRNA Differential Display 350
References 354
CHAPTER 17
Quantitative Polymerase Chain
Reaction Techniques
Rationale 357
Quantitative Approaches 357
Types of Internal Controls 358
Competitive PCR: Key Considerations 362
Competitive PCR: Major Steps Involved 368
Protocol: Competitive PCR 371
Image Analysis Considerations 377
Trouble Shooting Competitive PCR 378
Real Time PCR 380
References 383
Contents xv
CHAPTER 18
Quantification of Specific Messenger RNAs
by Nuclease Protection
Rationale 386
Basic Approach 388
Probe Selection 390
Optimization Suggestions 393
Potential Difficulties 395
Protocol: Transcript Quantification by SI Analysis 397
Protocol: Transcript Quantification by RNase Protection 400
References 404
CHAPTER 19
Analysis of Nuclear RNA
Introduction 407
Part I: Transcription Rate Assays 407
Rationale 407
Protocol: Nuclear Runoff Assay 413
Harvesting of Cells and Preparation of Nuclei 413
Cellular Lysis with NP 40 Buffer 415
Alternative Protocol for Preparation of Nuclei: Isolation of Fragile Nuclei
from Tissue Culture Cells 415
Alternative Protocol for Preparation of Nuclei: Isolation of Nuclei from
Whole Tissue 417
Labeling of Transcripts 418
Recovery of Labeled Transcripts 419
Preparation of Target DNA 421
Preparation of RNA for Hybridization 423
Posthybridization Washes and Detection 425
Protocol: Nuclear Runoff Assay: Alternative Procedure 425
Protocol: Nuclease Protection Pulse Label Transcription Assay 427
Distinguishing among the Activities of RNA Polymerases I, II, and
III 430
Part II: Extraction of Nuclear RNA for Steady State Analysis 431
Rationale 431
Protocol: Direct Isolation of Nuclear RNA 433
Protocol: Preparation of Nuclear RNA from Cells Enriched in
Ribonuclease 434
References 435
xvi Contents
CHAPTER 20
An RNA Paradigm
A Typical(?) Experiment 440
What to Do Next 444
EPILOGUE
A Few Pearls of Wisdom 447
APPENDIX A
Phenol Preparation
Phenol Saturation 454
Procedure 1: Tris Saturation 455
Procedure 2: Water Saturation 456
References 457
APPENDIX B
Disposal of Ethidium Bromide and SYBR
Green Solutions
Protocol 1 458
Ancillary Protocol: Quantitative Assay for Residual EtBr in Filtrate 459
Protocol 2 460
Protocol 3 460
References 461
APPENDIX C
DNase I Removal of DNA from an RNA Sample
Protocol 463
APPENDIX D
RNase Incubation to Remove RNA from a DNA Sample
Protocol: Removal of DNase Activity from RNase 464
Protocol: Digestion of RNA 465
References 465
Contents xvii
APPENDIX E
Deionization of Formamide, Formaldehyde,
and Glyoxal 466
APPENDIX F
Silanizing Centrifuge Tubes and Glassware
Protocol 1 467
Protocol 2 468
APPENDIX G
Centrifugation as a Mainstream Tool for the
Molecular Biologist
Types of Centrifuges 469
Rotors 470
Applications 471
Differential Centrifugation 472
Density Gradient Centrifugation—Sedimentation Velocity 472
Density Gradient Centrifugation—Isopycnic Technique 473
References 474
APPENDIX H
Trypsinization Protocol for Anchorage Dependent Cells
Protocol 475
APPENDIX I
Isolation of High Molecular Weight DNA by the
Salting Out Procedure
Protocol 478
References 481
APPENDIX J
RNA Isolation from Plant Tissue
References 482
wiii Contents
APPENDIX K
Electrophoresis: Principles, Parameters, and Safety
Theoretical Considerations 485
A Typical Electrophoretic Separation 487
Choice of Matrix 487
Purity of Agarose 489
Agarose Concentration 490
Polyacrylamide Concentration 490
Molecular Size Range of Sample 491
Nucleic Acid Concentration 491
Applied Voltage 491
Ethidium Bromide 492
SYBR Green 492
Base Composition and Temperature 493
Field Direction 493
Types of Gel Boxes 494
Safety Considerations in Electrophoresis 496
Maintenance of Electrophoresis Equipment 496
References 496
APPENDIX L
Polyacrylamide Gel Electrophoresis
Analytical 499
References 500
APPENDIX M
Internal Controls
References 503
APPENDIX N
Trade Citations 505
APPENDIX 0
Selected Suppliers of Equipment, Reagents,
and Services 507
Glossary 511
Index 525
|
| any_adam_object | 1 |
| author | Farrell, Robert E. Jr |
| author_facet | Farrell, Robert E. Jr |
| author_role | aut |
| author_sort | Farrell, Robert E. Jr |
| author_variant | r e f re ref |
| building | Verbundindex |
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| ctrlnum | (OCoLC)40166639 (DE-599)BVBBV012266375 |
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| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.886 |
| dewey-search | 572.886 |
| dewey-sort | 3572.886 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| edition | 2. ed. |
| format | Book |
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| id | DE-604.BV012266375 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T18:24:34Z |
| institution | BVB |
| isbn | 0122496957 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-008313637 |
| oclc_num | 40166639 |
| open_access_boolean | |
| owner | DE-20 DE-355 DE-BY-UBR DE-19 DE-BY-UBM DE-11 |
| owner_facet | DE-20 DE-355 DE-BY-UBR DE-19 DE-BY-UBM DE-11 |
| physical | XX, 533 S. Ill., graph. Darst. |
| publishDate | 1998 |
| publishDateSearch | 1998 |
| publishDateSort | 1998 |
| publisher | Acad. Press |
| record_format | marc |
| spelling | Farrell, Robert E. Jr. Verfasser aut RNA methodologies a laboratory guide for isolation and characterization Robert E. Farrell 2. ed. San Diego [u.a.] Acad. Press 1998 XX, 533 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier ARN - Analyse - Manuels de laboratoire Isolering (proces) gtt Karakterisering (algemeen) gtt RNA gtt Wetenschappelijke technieken gtt Wissenschaftliches Arbeiten Molecular Biology methods Laboratory Manuals RNA Analysis Laboratory manuals RNA isolation & purification Laboratory Manuals RNA physiology Laboratory Manuals Methode (DE-588)4038971-6 gnd rswk-swf Isolierung Chemie (DE-588)4122223-4 gnd rswk-swf RNS (DE-588)4076759-0 gnd rswk-swf Isolierung Mikrobiologie (DE-588)4193882-3 gnd rswk-swf Isolierung (DE-588)4027787-2 gnd rswk-swf RNS (DE-588)4076759-0 s Isolierung Mikrobiologie (DE-588)4193882-3 s DE-604 Isolierung Chemie (DE-588)4122223-4 s Isolierung (DE-588)4027787-2 s Methode (DE-588)4038971-6 s 1\p DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008313637&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Farrell, Robert E. Jr RNA methodologies a laboratory guide for isolation and characterization ARN - Analyse - Manuels de laboratoire Isolering (proces) gtt Karakterisering (algemeen) gtt RNA gtt Wetenschappelijke technieken gtt Wissenschaftliches Arbeiten Molecular Biology methods Laboratory Manuals RNA Analysis Laboratory manuals RNA isolation & purification Laboratory Manuals RNA physiology Laboratory Manuals Methode (DE-588)4038971-6 gnd Isolierung Chemie (DE-588)4122223-4 gnd RNS (DE-588)4076759-0 gnd Isolierung Mikrobiologie (DE-588)4193882-3 gnd Isolierung (DE-588)4027787-2 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4122223-4 (DE-588)4076759-0 (DE-588)4193882-3 (DE-588)4027787-2 |
| title | RNA methodologies a laboratory guide for isolation and characterization |
| title_auth | RNA methodologies a laboratory guide for isolation and characterization |
| title_exact_search | RNA methodologies a laboratory guide for isolation and characterization |
| title_full | RNA methodologies a laboratory guide for isolation and characterization Robert E. Farrell |
| title_fullStr | RNA methodologies a laboratory guide for isolation and characterization Robert E. Farrell |
| title_full_unstemmed | RNA methodologies a laboratory guide for isolation and characterization Robert E. Farrell |
| title_short | RNA methodologies |
| title_sort | rna methodologies a laboratory guide for isolation and characterization |
| title_sub | a laboratory guide for isolation and characterization |
| topic | ARN - Analyse - Manuels de laboratoire Isolering (proces) gtt Karakterisering (algemeen) gtt RNA gtt Wetenschappelijke technieken gtt Wissenschaftliches Arbeiten Molecular Biology methods Laboratory Manuals RNA Analysis Laboratory manuals RNA isolation & purification Laboratory Manuals RNA physiology Laboratory Manuals Methode (DE-588)4038971-6 gnd Isolierung Chemie (DE-588)4122223-4 gnd RNS (DE-588)4076759-0 gnd Isolierung Mikrobiologie (DE-588)4193882-3 gnd Isolierung (DE-588)4027787-2 gnd |
| topic_facet | ARN - Analyse - Manuels de laboratoire Isolering (proces) Karakterisering (algemeen) RNA Wetenschappelijke technieken Wissenschaftliches Arbeiten Molecular Biology methods Laboratory Manuals RNA Analysis Laboratory manuals RNA isolation & purification Laboratory Manuals RNA physiology Laboratory Manuals Methode Isolierung Chemie RNS Isolierung Mikrobiologie Isolierung |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008313637&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT farrellroberte rnamethodologiesalaboratoryguideforisolationandcharacterization |