RNA methodologies: a laboratory guide for isolation and characterization
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
Amsterdam [u.a.]
Elsevier, Acad. Press
2005
|
| Ausgabe: | 3. ed. |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XXV, 767 S. Ill., graph. Darst. |
| ISBN: | 0122496965 |
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_ | 1804133584578019328 |
|---|---|
| adam_text | Contents
Preface xxiii
CHAPTER 1
RNA and the Cellular Biochemistry Revisited
Why Study RNA? 2
What is RNA? 4
Assembly of Polynucleotides 7
Types of RNA 10
Stringency: Conditions That Influence Nucleic Acid Structure 11
The Effect of Salt on Stringency 13
The Effect of pH on Stringency 13
The Effect of Temperature on Stringency 14
The Effect of Formamide on Stringency 15
Types of Double Stranded Molecules 15
References and Suggested Reading 16
CHAPTER 2
Transcription and the Organization of Eukaryotic Genes
Transcription and the Central Dogma 18
Regulatory Elements 19
DNA Template 22
Gene Organization 22
vii
vjjj Contents
RNA Polymerases and the Products of Transcription 26
References 30
CHAPTER 3
Messenger RNA
Topology of a Typical mRNA Molecule 33
5 Cap 34
Leader Sequence 36
Coding Region 36
Trailer Sequence 37
Poly(A) Tail 37
Organellar mRNAs 39
Stability in the Cytoplasm 40
Levels of Regulation 40
References 43
CHAPTER 4
Resilient Ribonucleases
Rationale 48
Elimination of Ribonuclease Activity 48
Types of Ribonuclease Inhibitors 50
Specific Inhibitors 50
Nonspecific Inhibitors 52
Preparation of Equipment and Reagents 53
Diethyl Pyrocarbonate 54
Alternative: Sterile Water for Irrigation 57
Hydrogen Peroxide 57
Sodium Hydroxide and Sodium Dodecyl Sulfate 58
Other Reagents Used to Control Nuclease Activity 58
Guanidine Hydrochloride 59
Guanidine Thiocyanate 59
Sodium Dodecyl Sulfate 59
jV Laurylsarcosine 59
Phenol:Chloroform:Isoamyl Alcohol 60
8 Hydroxyquinoline 61
Cesium Chloride 61
Cesium Trifluoroacetate 61
Proteinase K 62
KNAlater 62
Protocol: Synthesis of Vanadyl Ribonucleoside Complexes 63
References 65
Contents ix
CHAPTER 5
RNA Isolation Strategies
Rationale 68
Goals in the Purification of RNA 69
Lysis Buffer Formulations 72
Gentle Lysis Buffers 73
RNA Isolation 77
Chaotropic Lysis Buffers 80
Isolation of RNA with Guanidinium Buffers 82
Guanidinium Acid Phenol Extraction Techniques 83
Protocol: Guanidinium Acid Phenol Extraction 85
Density Gradient Centrifugation 86
Cesium Chloride 87
Cesium Trifluoroacetate 93
Simultaneous Isolation of RNA and DNA 98
Protocol: Simultaneous Isolation of RNA and DNA 99
Recovery of RNA 101
Recovery of DNA 102
The Word on Kits 103
Silica Technology 103
Other Methods 104
Protocol: Rapid Isolation of RNA with Sodium Dodecyl Sulfate and Potassium
Acetate Reagents 105
Protocol: Isolation of Prokaryotic RNA 106
Protocol: Isolation of RNA from Yeast 108
Short and Long Term Storage of Purified RNA 110
References 112
CHAPTER 6
The Truth About Tissues
Rationale 115
Tissue Culture or Tissue? 115
Advantages of Cell Culture 116
Advantages of Tissue Samples 117
Homogenization Methods 117
Polytron Disruption 118
Dounce Homogenization 119
RNA Isolation Strategies for Various Organs and Tissues 120
Fresh Tissue 123
Frozen Tissue 123
Fixed Tissue 124
X Contents
Protocol: LiCl Urea Method for RNA Isolation from Tissue 125
Protocol: RNA Isolation from Lipid Enriched Tissue 129
Purification of Polysome Engaged mRNA 131
Protocol: Isolation of Polysomal mRNA 132
Collecting Samples in the Field 134
RNA Clean Up Methods 135
References 136
CHAPTER 7
Isolation of Polyadenylated RNA
Rationale 139
Polyadenylation 140
The Poly(A) Caveat 141
Selection of Polyadenylated Molecules 143
Magnetic Bead Technology for Poly(A)+ Purification 146
Protocol: Purification of Poly(A)+ RNA with Magnetic Beads 149
01igo(dT) Cellulose Column Chromatography 152
Protocol: Purification of Biophysical Quantities of Poly(A)+ RNA 153
Rapid, Non Column Poly(A)+ Purification 158
Protocol: Rapid, Non Column Poly(A)+ Purification 159
References 161
CHAPTER 8
Quality Control for RNA Preparations
Rationale 164
Quality Control Technique 1: Electrophoretic Profile of the RNA 164
Protocol 167
Quality Control Technique 2: Ultraviolet Spectrophotometry and
Absorption Ratios 169
Determination of Nucleic Acid Concentration and Purity 169
Quality Control Technique 3: Sample Capacity to Support RT PCR 176
Quality Control Technique 4: Northern Analysis 177
Quality Control Technique 5: Sample Capacity to Support In Vitro Translation 177
References 178
^ CHAPTER 9
Dot Blot Analysis
Rationale 180
Advantages and Disadvantages 182
Contents xi
Appropriate Positive and Negative Controls 184
Protocol: RNA Dot Blots 185
Protocol: DNA Dot Blots 187
Limitations of the Data 188
References 189
CHAPTER 10
Electrophoresis of RNA
Rationale I9l
Normalization of Nucleic Acids 192
Protocol: Poly(A) Normalization 195
RNA Denaturing Systems for Agarose Gel Electrophoresis 199
Formaldehyde Denaturation 201
Protocol: Formaldehyde Denaturing Gels 202
Glyoxal/Dimethyl Sulfoxide Denaturation 204
Protocol: Glyoxalation and Electrophoresis of RNA 206
Molecular Weight Standards 208
Proper Use of Standards 210
Ribosomal RNA 212
Gel Staining Techniques 220
Ethidium Bromide 220
SYBR Green 223
SYBRGold 225
GelStar 226
Silver Staining 226
Acridine Orange 226
Methylene Blue 227
Safety Considerations in Electrophoresis 229
Maintenance of Electrophoresis Equipment 229
Running Agarose Gels for the First Time: A Few Tips 230
Essential Vocabulary 230
Points to Keep in Mind 231
References 235
CHAPTER 11
Photodocumentation and Image Analysis
Rationale 239
Photodocumentation 239
Sample Visualization 241
Filtration 243
Safety First 244
xjj Contents
Tips for Optimizing Electrophoretograms 246
Inherent Limitations of Photographic and X Ray Films 250
Digital Image Analysis 251
Image Formats 257
Practical Considerations 258
Suggested Reading 260
CHAPTER 12
Northern Analysis
Rationale 262
Choice of Filter Membrane 263
Nitrocellulose 264
Nylon 265
Polyvinylidene Difluoride 266
Handling and Filter Preparation 266
Northern Transfer Techniques 267
Capillary Transfer 267
TurboBlotter 269
Vacuum Blotting 269
Positive Pressure 270
Electroblotting 271
Alkaline Blotting 272
Protocol: RNA Transfer by Passive Capillary Diffusion 272
Protocol: TurboBlotter Downward Transfer of RNA 275
Post Transfer Handling of Filters 279
Formaldehyde Denaturing Systems 279
Glyoxal Denaturing Systems 279
Immobilization Techniques 279
Baking 280
Crosslinking by Ultraviolet Irradiation 280
Protocol: Ultraviolet Crosslinking RNA to Nylon Filters 281
Postfixation Handling of Filters 282
References 283
^ CHAPTER 13
Nucleic Acid Probe Technology
Rationale 286
Probe Classification 287
Selection of Labeling System 288
Isotope Labeling 289
Nonisotopic Labeling 296
Contents xiii
DNA Probes 300
DNA Probe Synthesis 302
Antisense RNA Probes 306
Characteristics of RNA Probes 308
RNA Probe Synthesis 309
Probe Purification 311
Probe Storage 311
Internal Controls 312
References 315
CHAPTER 14
Practical Nucleic Acid Hybridization
Rationale 318
Factors Influencing Hybridization Kinetics and Specificity 319
Temperature 320
Ionic Strength 321
pH 321
Probe Length 321
Probe Concentration 322
G+C Content 322
Mismatching 323
Probe Complexity 324
Viscosity 324
Formamide 325
Hybridization Temperature 325
Tm for Long Probes 326
Tm for Oligonucleotide Probes 326
Hybridization and the Northern Analysis 327
Prehybridization: Filter Preparation 328
Protocol: Prehybridization (Long Probes) 328
Probe Denaturation 330
Hybridization 330
Posthybridization Stringency Washes 331
References 332
CHAPTER 15
Principles of Detection
Rationale 334
Autoradiography 335
Handling of Filter Membranes 339
X Ray Film 339
xiv Contents
Safelight 341
Exposure Time 341
Intensifying Screens 342
Fluorography 343
Preflashing Film 344
Type of Cassette 344
Development and Fixation 345
Autoradiography: Suggested Protocol 345
Nonisotopic Procedures 348
Biotin 349
Digoxigenin 351
Fluorescent Nucleotides 352
Direct Enzyme Labeling 352
Detection by Chemiluminescence 353
Chromogenic Detection Procedures 357
Digital Imaging Systems 358
References 359
CHAPTER 16
Quantification of Specific mRNAs by Nuclease Protection
Rationale 362
Basic Approach 365
Probe Selection 368
Optimization Suggestions 372
Potential Difficulties 374
Protocol: Transcript Quantification by SI Nuclease Assay 377
Protocol: Transcript Quantification by RNase Protection Assay 381
References 385
CHAPTER 17
Analysis of Nuclear RNA
Rationale 388
Transcription Rate Assays 388
Protocol: Nuclear Runoff Assay 395
Harvesting of Cells and Preparation of Nuclei 395
Cellular Lysis with NP 40 Buffer 396
Alternative Protocol for Preparation of Nuclei: Isolation of Fragile Nuclei from
Tissue Culture Cells 397
Alternative Protocol for Preparation of Nuclei: Isolation of Nuclei from
Whole Tissue 399
Labeling of Transcripts 399
Contents xv
Recovery of Labeled Transcripts 401
Preparation of Target DNA 403
Preparation of RNA for Hybridization 405
Posthybridization Washes and Detection 407
Protocol: Nuclear Runoff Assay: Alternative Procedure 407
Protocol: Nuclease Protection and Pulse Label Transcription Assay 409
Distinguishing Among the Activities of RNA Polymerases I, II, and III 412
Extraction of Nuclear RNA for Steady State Analysis 414
Protocol: Direct Isolation of Nuclear RNA 415
Protocol: Preparation of Nuclear RNA from Cells Enriched in Ribonuclease 417
References 418
CHAPTER 18
cDNA Synthesis
Rationale 422
cDNA Synthesis—An Overview 422
First Strand Considerations 424
Second Strand Considerations 433
Assessing Complementary DNA Synthesis Efficiency 436
Ligation Considerations 436
References 438
CHAPTER 19
RT PCR
Rationale 440
PCR—An Overview 440
RT PCR—General Approach 446
Laboratory Design 452
Primer Design 452
Basic Rules 458
T Considerations 461
m
Optimization Procedures 462
Analysis of PCR Products 471
RT PCR Quality Control Points 471
Related Techniques 474
5 RACE PCR 474
3 RACE PCR 477
Nested PCR 478
Protocol: First Strand cDNA Synthesis 479
Protocol: PCR Amplification of cDNA 481
Cloning PCR Products 482
xvi Contents
Protocol: A Tailing of Blunt End PCR Products 484
Protocol: TA Cloning Ligation Reaction 485
TOPO Cloning 487
References 487
CHAPTER 2 0
Quantitative PCR Techniques
Rationale 491
Sensitivity Index 492
Quantitative Approaches 493
Internal Controls 494
Exogenous Controls 497
Control Reaction Formats 500
Negative Control Considerations 503
Competitive PCR: Key Considerations 505
Competitive PCR: Major Steps Involved 512
Protocol: Competitive PCR 514
Synthesis of Nonhomologous Competitor 515
Synthesis of First Strand cDNA 517
Competitive PCR (Primary Amplification) 518
Competitive PCR (Secondary Amplification) 520
Image Analysis Considerations 522
Troubleshooting Competitive PCR 522
Real Time PCR 526
References 532
CHAPTER 2 1
Transcript Subtraction Methods
Rationale 537
Essential Issues 539
Subtraction Suppression PCR 541
Non PCR Subtraction 548
Troubleshooting Subtraction Methods 550
References 553
_^ CHAPTER 22
mRNA Differential Display
Rationale 556
General Approach 557
Contents xvii
Product Variety: What to Expect 568
Protocol: mRNA Differential Display 571
Synthesis of cDNA 571
Amplification of cDNA Subpopulations by PCR 573
PCR Product Analysis 574
Protocol: Identification and Selection of Differentially Expressed Sequences 577
Recovery of Differentially Expressed Sequences by Affinity Capture 578
Cloning PCR Products 580
Confirmation of Differential Expression 581
Subsequent Characterization 581
Applications of Differential Display 582
Troubleshooting mRNA Differential Display 583
References 586
CHAPTER 2 3
High Throughput Analysis of Gene Expression
Rationale 589
What Is a Microarray? 589
What Microarrays Can Do 591
What Microarrays Cannot Do 593
Major Steps in Microarray Analysis 596
Reference RNA 598
Applications 599
References 600
CHAPTER 2 4
RNA Interference:Targeted Gene Silencing
Rationale 602
Essential RNAi Nomenclature 603
RNAi—How It Works 605
siRNA Approach 608
shRNA Approach 608
DNA Directed RNA Interference 610
Short Interfering RNA Delivery Methods into Mammalian Cells 611
Effective Design of siRNAs 612
In Vitro and In Vivo Issues 614
References 617
xviii Contents
CHAPTER 2 5
Genomes, Transcriptomes, Proteomes, and Bioinformatics
Rationale 621
Essential Nomenclature 622
Genomes and Genomics 624
Transcriptomes and Transcriptomics 625
Proteomes and Proteomics 626
Bioinformatics 628
References 631
CHAPTER 2 6
An RNA Paradigm
A Typical Experiment? 635
Sensitivity Issues 639
What to Do Next 639
Where to Turn for Help 642
EPILOGUE
A Few Pearls of Wisdom 644
APPENDIX A
Maintaining Complete and Accurate Records 651
APPENDIX B
Useful Stock Solutions for the Molecular Biologist 654
^ APPENDIX C
Phenol Preparation 661
Phenol Saturation 662
Procedure 1: Tris Saturation 663
Procedure 2: Water Saturation 664
References 665
Contents xix
APPENDIX D
Disposal of Ethidium Bromide and SYBR
Green Solutions 666
Protocol 1 666
Ancillary Protocol 667
Protocol 2 668
Protocol 3 668
References 669
APPENDIX E
DNase I Removal of DNA from an RNA Sample 670
Protocol 671
APPENDIX F
RIMase Incubation to Remove RNA from a
DNA Sample 672
Protocol: Removal of DNase Activity from RNase 673
Protocol: Digestion of RNA 673
References 673
APPENDIX G
Deionization of Formamide, Formaldehyde, and Glyoxal 674
APPENDIX H
Silanizing Centrifuge Tubes and Glassware 676
Protocol 676
APPENDIX I
Centrifugation as a Mainstream Tool for the
Molecular Biologist 677
Types of Centrifuges 677
Rotors 678
xx Contents
Applications 681
Differential Centrifugation 681
Density Gradient Centrifugation—Sedimentation Velocity 681
Density Gradient Centrifugation—Isopycnic Technique 682
References 683
APPENDIX J
Trypsinization Protocol for Anchorage Dependent Cells 684
Protocol 684
APPENDIX K
Isolation of High Molecular Weight DNA by Salting Out 687
Protocol 688
References 690
APPENDIX L
RNA Isolation from Plant Tissue 691
APPENDIX M
Electrophoresis: Principles, Parameters, and Safety 693
Theoretical Considerations 694
A Typical Electrophoretic Separation 696
Choice of Matrix 696
Purity of Agarose 698
Agarose Concentration 699
Polyacrylamide Concentration 699
Molecular Size Range of Sample 700
Nucleic Acid Conformation 700
Applied Voltage 700
Ethidium Bromide 701
SYBR Green 702
Base Composition and Temperature 702
Field Direction 702
Types of Gel Boxes 703
Safety Considerations in Electrophoresis 704
Maintenance of Electrophoresis Equipment 705
References 705
Contents xxi
APPENDIX N
Polyacrylamide Gel Electrophoresis 707
Analytical 709
References 709
APPENDIX 0
Selected Suppliers of Equipment, Reagents,
and Services 710
APPENDIX P
Useful SI Units 716
APPENDIX Q
Common Abbreviations 718
APPENDIX R
Trademark Citations 721
Glossary 722
Index 739
|
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| id | DE-604.BV020021964 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T20:11:02Z |
| institution | BVB |
| isbn | 0122496965 |
| language | English |
| lccn | 2005041012 |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-013343323 |
| oclc_num | 57414698 |
| open_access_boolean | |
| owner | DE-20 DE-M49 DE-BY-TUM DE-578 |
| owner_facet | DE-20 DE-M49 DE-BY-TUM DE-578 |
| physical | XXV, 767 S. Ill., graph. Darst. |
| publishDate | 2005 |
| publishDateSearch | 2005 |
| publishDateSort | 2005 |
| publisher | Elsevier, Acad. Press |
| record_format | marc |
| spelling | Farrell, Robert E. Jr. Verfasser aut RNA methodologies a laboratory guide for isolation and characterization Robert E. Farrell 3. ed. Amsterdam [u.a.] Elsevier, Acad. Press 2005 XXV, 767 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier ARN - Analyse - Manuels de laboratoire Laboratoriumonderzoek gtt RNA gtt RNA Analysis Laboratory manuals RNS (DE-588)4076759-0 gnd rswk-swf Isolierung Chemie (DE-588)4122223-4 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Isolierung (DE-588)4027787-2 gnd rswk-swf Isolierung Mikrobiologie (DE-588)4193882-3 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=013343323&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 Laboratoriumonderzoek gtt RNA gtt RNA Analysis Laboratory manuals RNS (DE-588)4076759-0 gnd Isolierung Chemie (DE-588)4122223-4 gnd Methode (DE-588)4038971-6 gnd Isolierung (DE-588)4027787-2 gnd Isolierung Mikrobiologie (DE-588)4193882-3 gnd |
| subject_GND | (DE-588)4076759-0 (DE-588)4122223-4 (DE-588)4038971-6 (DE-588)4027787-2 (DE-588)4193882-3 |
| 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 Laboratoriumonderzoek gtt RNA gtt RNA Analysis Laboratory manuals RNS (DE-588)4076759-0 gnd Isolierung Chemie (DE-588)4122223-4 gnd Methode (DE-588)4038971-6 gnd Isolierung (DE-588)4027787-2 gnd Isolierung Mikrobiologie (DE-588)4193882-3 gnd |
| topic_facet | ARN - Analyse - Manuels de laboratoire Laboratoriumonderzoek RNA RNA Analysis Laboratory manuals RNS Isolierung Chemie Methode Isolierung Isolierung Mikrobiologie |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=013343323&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT farrellroberte rnamethodologiesalaboratoryguideforisolationandcharacterization |