RNA methodologies: laboratory guide for isolation and characterization
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
London ; San Diego, CA ; Cambridge, MA ; Oxford
Academic Press, an imprint of Elsevier
[2017]
|
| Ausgabe: | Fifth edition |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Literaturangaben |
| Beschreibung: | xx, 855 Seiten Illustrationen, Diagramme |
| ISBN: | 9780128046784 |
Internformat
MARC
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| 100 | 1 | |a Farrell, Robert E. |e Verfasser |0 (DE-588)1144509459 |4 aut | |
| 245 | 1 | 0 | |a RNA methodologies |b laboratory guide for isolation and characterization |c Robert E. Farrell, Jr. |
| 250 | |a Fifth edition | ||
| 264 | 1 | |a London ; San Diego, CA ; Cambridge, MA ; Oxford |b Academic Press, an imprint of Elsevier |c [2017] | |
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Datensatz im Suchindex
| _version_ | 1804177892535435264 |
|---|---|
| adam_text | Contents
Preface xvii
1. RNA and the Cellular Biochemistry Revisited
Why Study RNA? 1
What is RNA? 2
Polynucleotide Synthesis 5
Types of RNA 9
Transcription and the Central Dogma 11
Promoters, Transcription Factors, and Regulatory Elements 13
Gene and Genome Organization Affect Transcription 18
RNA Polymerases and the Products of Transcription 23
Hallmarks of a Typical mRNA 27
5 Cap 29
5 UTR (Leader Sequence) 30
Coding Region 31
3 UTR (Trailer Sequence) 32
Poly(A) Tail 32
Organeliar mRNAs 34
mRNA Stability and Turnover 35
Bicistronic mRNAs 38
Prokaryotic mRNAs 38
mRNA Sequence and Structure Affect Translation 39
Levels of Gene Regulation 41
Alternative Splicing of mRNA from a Single Genetic Locus 43
Trans-Splicing: mRNA Repair 44
Overview of Noncoding RNAs 46
References 47
Further Reading 53
2. Creating a Ribonuclease-Free Environment
Rationale 55
Elimination of Resilient Ribonucleases 56
Latent RNase Contamination Issues 58
Types of Ribonucléase Inhibitors 59
Specific inhibitors 60
Nonspecific Inhibitors 62
V
vi Contents
Preparation of Equipment and Reagents 63
Diethyl Pyrocarbonate (DEPC) 64
Alternative: USP Sterile Water 66
Hydrogen Peroxide - 66
NaOH and SDS 67
Other Reagents Used to Control Nuclease Activity 67
Guanidinium Salts 67
Sodium Dodecyl Sulfate 68
N-Laurylsarcosine 68
Phenol:Chioroform:l$oamy! Alcohol 68
8-Hydroxyquinoline 69
Cesium Chloride 70
Cesium Trifluoroacetate 70
Proteinase K 70
RNA later 71
Protocol: Synthesis of VDR 71
References 73
3. RNA Isolation Strategies
Rationale 75
Goals in the Purification of RNA 77
Lysis Buffer Formulations 80
Gentle Lysis Buffers 80
Chaotropic Lysis Buffers 87
Isolation of RNA with Guanidinium Buffers 88
Guanidinium-Acid-Phenol Extraction Techniques 89
Protocol: Guanidinium-Acid-Phenol Extraction 91
Density Gradient Centrifugation 92
Cesium Chloride 93
Cesium Trifluoracetate (CsTFA) 97
Isolation of RNA and DNA from the Same Source 101
Protocol: Simultaneous Isolation of RNA and DNA 101
The Word on Kits 104
Silica Technology 105
Isolation of Cytoplasmic RNA on a Silica Column 106
Affinity Matrices 107
Other Methods 107
Protocol: Rapid Isolation of RNA with SDS and
Potassium Acetate Reagents 108
Protocol: Isolation of Prokaryotic RNA 109
Protocol: Isolation of RNA From Yeast 110
Short- and Long-Term Storage of Purified RNA 112
References 114
4. The Truth About Tissues
Rationale 117
Tissue Culture or Tissue? 117
Contents vii
Advantages of Cell Culture 118
Advantages of Tissue Samples 119
Homogenization Methods 120
Motorized Homogenizes 120
Dounce Homogenization 121
BeadBeater Homogenization 123
RNA Isolation Strategies for Various Organs and Tissues 125
Fresh Tissue 126
Frozen Tissue 127
Fixed Tissue 128
Protocol: LiCI-Urea Method for RNA Isolation From Tissue 130
Protocol: RNA Isolation From Lipid-Enriched Samples 133
Purification of Polysome- and Protein-Engaged mRNA 135
Protocol: Isolation of Polysomal mRNA 137
Collecting Samples in the Field 139
RNA Clean-Up Methods 140
Troubleshooting RNA Isolation From Tissue 142
References 143
Further Reading 144
5. Going Green: RNA and the Molecular Biology of Plants
Rationale 145
RNA Isolation and the Peculiarities of Plants 145
Types of RNA Produced in Plant Cells 149
Protocol: RNA Isolation From Leaf 151
Protocol: RNA Isolation From Bark 153
Protocol: RNA Isolation From Fruit 155
Protocol: RNA Isolation From Plant Tissue With Hot Borate 158
Strategies for RNA Isolation From Other Plant Tissues 160
Troubleshooting RNA isolation From Plant Tissue 161
References 162
Further Reading 164
6. Quality Control for RNA Preparations
Rationale 167
Quality Control Technique 1: UV Spectrophotometry
and Absorption Ratios 167
Determination of Nucleic Acid Concentration 168
Determination of Nucleic Acid Purity 171
Nonspectrophotometric Methods 174
Quality Control Technique 2: Electrophoretic Profiling of RNA 176
Protocol: Nondenaturing Agarose Electrophoresis 179
Quality Control Technique 3: RNA Integrity Number (RIN) 180
Quality Control Technique 4: UV Shadowing 182
Protocol: UV Shadowing 182
Quality Control Technique 5: Sample Capacity to Support RT-PCR 184
184
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250
Contents
Quality Control Technique 6: Sample Capacity to
Support Northern Analysis
Quality Control Technique 7: Sample Capacity to
Support In Vitro Translation
References
cDNA—A Permanent Biochemical Record of the Cell
Rationale
cDNA Synthesis—An Overview
First-Strand Considerations
Reverse Transcriptase Options
Second-Strand Considerations
Classical Methods
PCR-Based Methods
Protocol: First-Strand cDNA Synthesis
Assessing cDNA Synthesis Efficiency
Cloning cDNA
Ligation Considerations
Enzymes Used for Ligation
Applications
References
RT-PCR: A Science and an Art Form
Rationale
PCR—An Overview
RT-PCR— General Approach
PCR Carryover Prevention
Laboratory Design
Procedural Methods
Aerosol-Resistant Tips
Uracil-N-GIycosylase
Primer Design
Basic Rules
Tm Considerations
AG Considerations
Multiplex Primer Design
Optimization Procedures
Thermostable Polymerases
Positive Controls
Negative Controls
Hot-Start PCR
Locked Nucleic Acid
Touchdown PCR
Internal Controls
The Word on Transcription Controls
Analysis of PCR Products
Contents IX
RT-PCR Quality Control Points 251
Non-PCR Methods for Confirming PCR-Derived Data 254
Related Techniques 255
5 RACE PCR 255
5 RLM-RACE 256
3 RACE PCR 258
Nested PCR 259
Long-Range PCR 260
Single-Cel! PCR 263
Splinkerette PCR 264
The Hunt for Alternative Transcription Start Sites 265
Protocol: First-Strand cDNA Synthesis 266
Protocol: PCR Amplification of cDNA 267
Cloning PCR Products 268
Protocof: A-Taiiing of Biunt-End PCR Products 270
Protocol: TA Cloning Ligation Reaction 270
TOPO Cloning 272
Other Amplification Procedures 272
Linear RNA Amplification (Eberwine Process) 273
Strand Displacement Amplification 274
Nucleic Acid Sequence Based Amplification (NASBA) 275
Roiling Circle Amplification (RCA) 275
Ligase Chain Reaction 275
LAMP 276
References and Suggested Reading 276
9. Quantitative PCR Techniques
Rationale 283
Sensitivity Index 284
Quantitative Approaches 285
The MIQE Guidelines 287
Real-time PCR 288
Real-Time PCR Platforms 292
SYBR Green Assay 293
TaqMan Assay 294
Molecular Beacons 295
Scorpions 297
Melting Curve Analysis 298
Digital PCR 300
Internal Controls 302
Exogenous Controls 302
Control Reaction Formats 304
Negative Control Considerations 308
PCR Arrays 310
Competitive PCR: Key Considerations 311
Competitive PCR: Major Steps Involved 316
Alternative Approach: Nonreal-Time Competitive PCR 318
x Contents
Protocol: Competitive PCR 318
Troubleshooting Quantitative PCR Techniques 324
References 327
10. miRNA
Rationale 329
Structural and Functional Characteristics of miRNA 329
miRNA Biogenesis 331
Differences and Similarities of miRNA and siRNA 335
miRNA Profiling 336
miRNA as Key Regulator of Gene Expression 338
miRNA Biomarkers 340
References and Suggested Reading 342
11. RNA Interference and RNA Editing
Rationale 345
Essential RNAi Terminology 346
RNA Interference —How It Works 348
Endogenous Silencing Pathways 351
Exogenous Silencing Strategies 353
Effective Design of siRNAs 359
RNAi and Alternative Transcript Splicing 363
In Vitro and In Vivo Issues 363
RNAi Validation 367
RT-PCR Approaches 367
Northern Analysis 367
Western Analysis and Other Protein Methods 368
RNAi Applications 368
CRISPR—Cas9 369
References 373
Further Reading 376
12. Stringency: Conditions That Influence Nucleic Acid
Structure
Rationale 377
Types of Double-Stranded Molecules 377
importance of Controlling Stringency 378
Effect of Salt on Stringency 380
Effect of pH on Stringency 380
Effect of Temperature on Stringency 381
Effect of Formamide on Stringency 381
Effect of Urea on Stringency 382
References 382
Contents xi
13. Electrophoresis of RNA
Rationale 383
Normalization of Samples by Nucleic Acid Concentration 384
Direct Measurement of Poly(A) Content 385
Protocol: Poly(A) Normalization With a Poly(T) Probe 387
Intramolecular Base-Pairing Mandates RNA Dénaturation 390
Formaldehyde Dénaturation 392
Protocol: Formaldehyde-Denaturing Gels 393
Urea Dénaturation 395
Protocol: Urea Dénaturation 395
Glyoxal/Dimethyl Sulfoxide Dénaturation 396
Protocol: Glyoxalation and Electrophoresis of RNA 397
Gel and Sample Preparation 398
Running RNA on Nondenaturing Gels 399
Molecular Weight Standards 400
Proper Use of Size Standards 401
Ribosomal RNA 402
Cel Staining Options 410
Ethidium Bromide 410
SVBR Green 413
SYBR Gold 413
SYBR Safe 415
GelStar 416
Stiver Staining 416
Acridine Orange 416
Methylene Blue 417
Safety Considerations and Equipment Maintenance 418
A Few Tips for Running Agarose Gels for the First Time 420
References 424
14. Photodocumentation and Image Analysis
Rationale 427
Safety First 427
Digital Image Analysis 428
Image Enhancement 431
Filtration 434
Image Formats 436
Practical Considerations 436
Image Analysts Training 439
Phosphorlmagers 440
Traditional Methods of Photodocumentation 440
Camera Settings 441
Tips for Optimizing Electrophoretograms 442
Inherent Limitations of Photographic and X-ray Films 445
References and Suggested Reading 446
xii Contents
15. Northern Analysis
Rationale 447
Choice of Blotting Membrane 448
Nitrocellulose 449
Nylon 450
Polyvinylidene Difluoride 451
Handling and Membrane Preparation 451
Northern Transfer Techniques 452
Capillary Transfer 452
TurboBlotter 454
Vacuum Blotting 454
Electroblotting 455
Alkaline Blotting 456
Protocol: RNA Transfer by Passive Capillary Diffusion 457
Protocol: Turboblotter Downward Transfer of RNA 459
Immobilization Techniques 462
Baking 463
Crosslinking by UV irradiation 463
Protocol: UV Crosslinking RNA to Nylon Filters 464
Postfixation Handling of Blotting Membranes 465
Reverse Northern Analysis 466
References 467
16. Nucleic Add Probe Technology
Rationale 469
DNA Probes 470
DNA Probe Synthesis 471
Sense and Antisense RNA Probes 475
RNA Probe Synthesis 477
Selection of Labeling System 479
Isotope Labeling 479
Popular Chemiluminescence Platforms 482
Direct Enzyme Labeling 483
Biotin 484
Digoxigenin 486
Fluorescein 487
The Ubiquitous Dyes Cy3 and Cy5 487
Probe Purification 488
Probe Storage 489
Factors Influencing Hybridization Kinetics and Duplex Stability 490
Temperature and Tm Considerations 491
tonic Strength 494
pH 494
Probe Length 494
Probe Concentration 494
G -f C Content 495
Contents xiii
Mismatching 496
Probe Complexity 497
Example 497
Viscosity 497
Formamide 498
Urea 498
Mixed-Phase Hybridization; Northern and Southern Blots 499
Prehybridization: Filter Preparation 499
Protocol: Prehybridization for Long Probes 500
Protocol: Generic Method for Probe Removal 503
Principles of Detection 505
Digital Imaging Systems 505
Autoradiography 506
Non isotopic Procedures 517
Biotin 518
Digoxigenin 518
Fluorescein and Fluorescence Imaging 519
Direct Enzyme Labeling 519
Detection by Chemiluminescence 519
Substrates for Chemiluminescence 521
Chromogenie Detection Procedures 523
References 524
17. Isolation of Polyadenylated RNA
Rationale 527
Polyadenylation 528
Selection of Polyadenylated Molecules: How it Works 529
The Poly(A) Caveat 531
Example 1 - cDNA Synthesis Considerations 531
Example 2 - Assay Sensitivity Considerations 532
Magnetic Bead Technology for Poly(A)+ Purification 533
Oligo(dT)-Ce)lulose Chromatography 535
Protocol: Noncolumn PoIy(A)+ Purification 536
Protocol: Large-Scale Poly(A)+ RNA Purification with
Oiigo(dT)-CelIulose Beads 538
References 543
18. Quantification of Specific mRNAs by
Nuclease Protection
Rationale 545
Basic Approach 546
Probe Selection 551
Optimization Suggestions 555
Potential Difficulties 556
Protocol: Transcript Quantification by S1 Analysis 558
xiv Contents
Protocol: Transcript Quantification by RNase Protection 562
Troubleshooting 565
References 566
19. Analysis of Nuclear RNA
Rationale 569
Transcription Rate Assays 569
Relationship to the Study of Steady-State RNA 574
Nuclear Run-off Versus Nuclear Run-on Assay 576
Protocol: Nuclear Run-on Assay 577
Harvesting of Cells and Preparation of Nuclei 577
Alternative Protocol for Preparation of Nuclei From Cell Culture 578
Alternative Protocol for Preparation of Nuclei From Whole Tissue 580
Labeling and Recovery of Transcripts 581
Preparation of Target DNA 583
Preparation of RNA for Hybridization 585
Posthybridization Washes and Detection 586
Protocol: Alternative Procedure for Nuclear Run-on Assay 587
Protocol: Nuclease Protection—Pulse Label Transcription Assay 588
Distinguishing Among the Activities of RNA Polymerases 591
Extraction of Nuclear RNA for Steady-State Analysis 592
Protocol: Direct Isolation of Nuclear RNA 593
Protocol: Preparation of Nuclear RNA From Cells Enriched in
Ribonucléase 595
Troubleshooting Nuclear RNA Analysis 596
References 597
Further Reading 599
20. Nonarray Methods for Global Analysis of
Gene Expression
Rationale 601
Essential Issues 602
Subtractive Methods 603
Suppression Subtractive Hybridization (SSH) 603
Troubleshooting 611
Nonsubtractive Methods 613
mRNA Differential Display 614
Troubleshooting 623
References 625
21. Genomes, Transcriptomes, Proteomes,
and Bioinformatics
Rationale 629
Essential Nomenclature 631
Transcriptomes and Transcriptomics 632
RNA-Seq: The Premier Transcriptomics Tool 633
Contents XV
Genomes and Genomics 639
Proteomes and Proteomics 641
RNA—RNA and RNA—Protein Interactions 646
Bioinformatics 648
Search for Genes —Have a BLAST! 649
References 652
22* RNA Biomarker Discovery
Rationale 655
Biomarkers Defined 655
Characteristics of Useful Biomarkers 657
Circulating RNA 658
Identification of Biomarkers for Research and Diagnostic
Applications 659
DNA Approaches 660
RNA Approaches 661
Protein Approaches 663
Metabolomics Approaches 664
Biomarker Issues and Shortcomings 664
References and Suggested Reading 665
23. High-Throughput Analysis of Gene Expression
Rationale 667
What is a Microarray? 667
What Is a Heat Map? 672
What Microarrays Can Do 674
What Microarrays Cannot Do 674
Major Steps in Microarray Analysis 677
Reference RNA 679
What Is a Macroarray? 680
Applications 682
References 684
24. Functional Genomics and Transcript Profiling
Rationale 685
Functional Genomics Defined 686
importance of Functional Genomics Approaches 686
Commonly Used Functional Genomics Approaches 687
Relationship of Functional Genomics Approaches
to Classical Molecular Biology 693
References 695
Further Reading 695
xvi Contents
25. A Few RNA Success Stories
A Typical Experiment? 698
Sensitivity Issues 701
What to Do Next 701
Where to Turn for Help 703
Current Innovations 704
RNA Biobanking 704
RNA Reprogramming 705
References 706
Epilogue: A Few Pearls of Wisdom 707
Appendix A: Maintaining Complete and Accurate Records 715
Appendix B: Converting Mass to Moles 719
Appendix C: Disposal of Ethidium Bromide and SYBR Green Solutions 723
Appendix D: Removal of DNA From an RNA Sample 727
Appendix E: Removal of RNA From a DNA Sample 729
Appendix F: Useful Stock Solutions for the Molecular Biologist 731
Appendix G: Silanizing Centrifuge Tubes and Glassware 737
Appendix H: Dot Blot Analysis 739
Appendix I: Electrophoresis: Principles, Parameters, and Safety 749
Appendix J: Polyacrylamide Gel Electrophoresis 761
Appendix K: Centrifugation as a Mainstream Tool for the Molecular
Biologist 765
Appendix L: Trypsinization Protocol for Anchorage-Dependent Cells 773
Appendix M: Isolation of High-Moiecuiar-Weight DNA by Salting-Out 777
Appendix N: Phenol Preparation 781
Appendix O: Deionization of Formamide, Formaldehyde, and Glyoxal 785
Appendix P: Selected Suppliers of Equipment, Reagents, and Services 787
Appendix Q: Useful SI Prefixes and Common SI Base Units 797
Appendix R: Common Abbreviations 799
Appendix S: Trademark Citations 801
Glossary 805
Index 833
|
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| id | DE-604.BV044536837 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T07:55:18Z |
| institution | BVB |
| isbn | 9780128046784 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-029935987 |
| oclc_num | 1011128489 |
| open_access_boolean | |
| owner | DE-29T DE-355 DE-BY-UBR DE-11 DE-19 DE-BY-UBM DE-20 |
| owner_facet | DE-29T DE-355 DE-BY-UBR DE-11 DE-19 DE-BY-UBM DE-20 |
| physical | xx, 855 Seiten Illustrationen, Diagramme |
| publishDate | 2017 |
| publishDateSearch | 2017 |
| publishDateSort | 2017 |
| publisher | Academic Press, an imprint of Elsevier |
| record_format | marc |
| spelling | Farrell, Robert E. Verfasser (DE-588)1144509459 aut RNA methodologies laboratory guide for isolation and characterization Robert E. Farrell, Jr. Fifth edition London ; San Diego, CA ; Cambridge, MA ; Oxford Academic Press, an imprint of Elsevier [2017] xx, 855 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Literaturangaben ARN - Analyse - Manuels de laboratoire Methode (DE-588)4038971-6 gnd rswk-swf Isolierung (DE-588)4027787-2 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 RNS (DE-588)4076759-0 s Isolierung Mikrobiologie (DE-588)4193882-3 s DE-604 Isolierung (DE-588)4027787-2 s Methode (DE-588)4038971-6 s Isolierung Chemie (DE-588)4122223-4 s 1\p DE-604 Digitalisierung UB Regensburg - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029935987&sequence=000001&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. RNA methodologies laboratory guide for isolation and characterization ARN - Analyse - Manuels de laboratoire Methode (DE-588)4038971-6 gnd Isolierung (DE-588)4027787-2 gnd Isolierung Chemie (DE-588)4122223-4 gnd RNS (DE-588)4076759-0 gnd Isolierung Mikrobiologie (DE-588)4193882-3 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4027787-2 (DE-588)4122223-4 (DE-588)4076759-0 (DE-588)4193882-3 |
| title | RNA methodologies laboratory guide for isolation and characterization |
| title_auth | RNA methodologies laboratory guide for isolation and characterization |
| title_exact_search | RNA methodologies laboratory guide for isolation and characterization |
| title_full | RNA methodologies laboratory guide for isolation and characterization Robert E. Farrell, Jr. |
| title_fullStr | RNA methodologies laboratory guide for isolation and characterization Robert E. Farrell, Jr. |
| title_full_unstemmed | RNA methodologies laboratory guide for isolation and characterization Robert E. Farrell, Jr. |
| title_short | RNA methodologies |
| title_sort | rna methodologies laboratory guide for isolation and characterization |
| title_sub | laboratory guide for isolation and characterization |
| topic | ARN - Analyse - Manuels de laboratoire Methode (DE-588)4038971-6 gnd Isolierung (DE-588)4027787-2 gnd Isolierung Chemie (DE-588)4122223-4 gnd RNS (DE-588)4076759-0 gnd Isolierung Mikrobiologie (DE-588)4193882-3 gnd |
| topic_facet | ARN - Analyse - Manuels de laboratoire Methode Isolierung Isolierung Chemie RNS Isolierung Mikrobiologie |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029935987&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT farrellroberte rnamethodologieslaboratoryguideforisolationandcharacterization |