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RNA methodologies : a laboratory guide for isolation and characterization / Robert E. Farrell, Jr. — 4th ed. — Amsterdam : Elsevier, c2010. – (58.174251/F245/4th ed.) |
Contents
Contents
1 RNA and the Cellular Biochemistry Revisited
Why Study RNA?
What is RNA?
Types of RNA
Transcription and the Central Dogma
Important Plant and Animal Models for Studying Transcription
Promoters and Regulatory Elements
Gene and Genome Organization Affect Transcription
RNA Polymerases and the Products of Transcription
Messenger RNA
Levels of Gene Regulation
Alternative Splicing of mRNA from a Single Genetic Locus
Trans-splicing: mRNA Repair
Overview of Small RNAs
miRNA Regulation of Gene Expression
References
2 RNA Isolation Strategies
Rationale
Goals in the Purification of RNA
Lysis Buffer Formulations
Isolation of RNA with Guanidinium Buffers
Guanidinium-Acid-Phenol Extraction Techniques
Density Gradient Centrifugation
Simultaneous Isolation of RNA and DNA
The Word on Kits
Other Methods
Short- and Long-Term Storage of Purified RNA
References
3 The Truth about Tissues
Rationale
Tissue Culture or Tissue?
Homogenization Methods
RNA Isolation Strategies for Various Organs and Tissues
Collecting Samples in the Field
RNA "Clean-Up" Methods
Troubleshooting RNA Isolation from Tissue
References
4 Going Green: RNA and the Molecular Biology of Plants
Rationale
RNA Isolation and the Peculiarities of Plants
Types of RNA Produced in Plant Cells
Protocol: RNA Isolation from Leaf
Protocol: RNA Isolation from Bark
Protocol: RNA Isolation from Fruit
Strategies for RNA Isolation from Other Plant Tissues
Troubleshooting RNA Isolation from Plant Tissue
References and Suggested Reading
5 Isolation of Polyadenylated RNA
Rationale
Polyadenylation
The Poly(A) Caveat
Selection of Polyadenylated Molecules
Magnetic Bead Technology for Poly(A)~ Purification
Oligo(dT)-Cellulose Column Chromatography
References
6 Quality Control for RNA Preparations
Rationale
Quality Control Technique 1: UV Spectrophotometry and Absorption Ratios
Quality Control Technique 2: Electrophoretic Profile of the RNA
Quality Control Technique 3: UV Shadowing
Quality Control Technique 4: Sample Capacity to Support RT-PCR
Quality Control Technique 5: Northern Analysis
Quality Control Technique 6: Sample Capacity to Support
In Vitro Translation
References
7 Resilient Ribonucleases
Rationale
Elimination of Ribonuclease Activity
Latent RNase Contamination Issues
Types of Ribonuclease Inhibitors
Preparation of Equipment and Reagents
Other Reagents Used to Control Nuclease Activity
Protocol: Synthesis of VDR
References
8 Stringency: Conditions that Influence Nucleic Acid Structure
Types of Double-Stranded Molecules
Importance of Controlling Stringency
Effect of Urea on Stringency
References
9 Electrophoresis of RNA
Rationale
Normalization of Nucleic Acids
RNA Denaturing Systems for Agarose Gel Electrophoresis
Urea Denaturation
Running RNA on Non-denaturing Gels
Molecular Weight Standards
Gel Staining Techniques
Safety Considerations and Equipment Maintenance
Running Agarose Gels for the First Time: A Few Tips
References
10 Photodocumentation and Image Analysis
Rationale
Safety First
Digital Image Analysis
Traditional Methods of Photodocumentation
References and Suggested Reading
11 Northern Analysis
Rationale
Choice of Filter Membrane
Handling and Filter Preparation
Northern Transfer Techniques
Post-Transfer Handling of Filters
Immobilization Techniques
Post-Fixation Handling of Filters
Reverse Northern Analysis
References
12 Nucleic Acid Probe Technology
Rationale
Probe Classification
Selection of Labeling System
Popular Chemiluminescence Formats
DNA Probes
Antisense RNA Probes
Probe Purification
Probe Storage
References
13 Practical Nucleic Acid Hybridization
Rationale
Factors Influencing Hybridization Kinetics and Duplex Stability
Hybridization Temperature
Hybridization and the Northern Analysis
Probe Removal and Rehybridization
References
14 Principles of Detection
Rationale
Autoradiography
Protocol: Autoradiography
Non-isotopic Procedures
Digital Imaging Systems
References
15 Quantification of Specific mRNAs by Nuclease Protection
Rationale
Basic Approach
Probe Selection
Optimization Suggestions
Potential Difficulties
Protocol: Transcript Quantification by S1Analysis
Protocol: Transcript Quantification by RNase Protection
Troubleshooting
References
16 Analysis of Nuclear RNA
Rationale
Transcription Rate Assays
Protocol: Nuclear Run-off Assay
Protocol: Alternative Procedure for Nuclear Run-off Assay
Protocol: Nuclease Protection - Pulse Label Transcription Assay
Distinguishing Among the Activities of RNA Polymerases
Extraction of Nuclear RNA for Steady-State Analysis
Protocol: Direct Isolation of Nuclear RNA
Troubleshoot Nuclear RNA Analysis
References
17 cDNA Synthesis
Rationale
cDNA Synthesis - An Overview
Protocol: first-strand cDNA Synthesis
Assessing cDNA Synthesis Efficiency
Cloning cDNA
Applications
References
18 RT-PCR: A Science and an Art Form
Rationale
PCR - An Overview
RT-PCR- General Approach
PCR Carryover Prevention
Primer Design
Optimization Procedures
Internal Controls
The Word on Transcription Controls
Analysis of PCR Products
RT-PCR Quality Control Points
Non-PCR Methods for Confirming PCR-derived Data
The Hunt for Alternative Transcription Start Sites
Protocol: First-Strand cDNA Synthesis
Protocol: PCR Amplification of cDNA
Cloning PCR Products
Other Amplification Procedures
References
19 Quantitative PCR Techniques
Rationale
Sensitivity Index
Quantitative Approaches
Real-Time PCR
Internal Controls
Exogenous Controls
Control Reaction Formats
Negative Control Considerations
Competitive PCR: Key Considerations
Competitive PCR: Major Steps Involved
Alternative Approach: Non-Real-Time Competitive PCR
References
20 Functional Genomics and Transcript Profiling
Rationale
Functional Genomics Defined
Importance of Functional Genomics Approaches
Commonly Used Functional Genomics Approaches
Functional Genomics and Classical Molecular Biology
21 High-Throughput Analysis of Gene Expression
Rationale
What is a Microarray?
What is a Heat Map?
What Microarrays Can Do
What Microarrays Cannot Do
Major Steps in Microarray Analysis
Reference RNA
What is a Macroarray?
Applications
References
22 Non-Array Methods for Global Analysis of Gene Expression
Rationale
Essential Issues
Non-Subtractive Methods
References
23 RNAi: Take a RISC-Role the Dicer
Rationale
Essential RNAi Nomenclature
RNA Interference - How it Works
Effective Design of siRNAs
RNAi and Alternative Transcript Splicing
RNAi Validation
Applications
References
24 Genomes， Transcriptomes， Proteomes， and Bioinformatics
Rationale
Essential Nomenclature
Genomes and Genomics
Transcriptomes and Transcriptomics
Proteomes and Proteomics
Bioinformatics
Search for Genes - Have a BLAST!
References
25 An RNA Paradigm
A Typical Experiment?
Sensitivity Issues
What to Do Next
Where to Turn for Help
EPILOGUE
A Few Pearls of Wisdom
Appendix A Maintaining Complete and Accurate Records
Appendix B Converting Mass to Moles
Appendix C Useful Stock Solutions for the Molecular Biologist
Appendix D Phenol Preparation
Appendix E Disposal of Ethidium Bromide and SYBR Green Solutions
Appendix F DNase I Removal of DNA from an RNA Sample
Appendix G RNase Incubation to Remove RNA from a DNA Sample
Appendix H Deionization of Formamide， Formaldehyde， and Glyoxal
Appendix I Silanizing Centrifuge Tubes and Glassware
Appendix J Trypsinization Protocol for Anchorage-Dependent Cells
Appendix K Isolation of High-Molecular-Weight DNA by Salting-Out
Appendix L Electrophoresis: Principles， Parameters， and Safety
Appendix M Polyacrylamide Gel Electrophoresis
Appendix N Dot Blot Analysis
Appendix O Centrifugation as a Mainstream Tool for the Molecular Biologist
Appendix P Selected Suppliers of Equipment， Reagents， and Services
Appendix Q Useful SI Units
Appendix R Common Abbreviations
Appendix S Trademark Citations
GLOSSARY
INDEX
