Dedication
Preface
Chapter 1: RNA and the Cellular Biochemistry Revisited
Why Study RNA?
What Is RNA?
Assembly of Polynucleotides
Types of RNA
Stringency: Conditions That Influence Nucleic Acid Structure
Types of Double-Stranded Molecules
Chapter 2: Transcription and the Organization of Eukaryotic Genes
Transcription and the Central Dogma
Gene Organization
RNA Polymerases and the Products of Transcription
Chapter 3: Messenger RNA
Topology of a Typical mRNA Molecule
Stability in the Cytoplasm
Levels of Regulation
Chapter 4: Resilient Ribonucleases
Rationale
Elimination of Ribonuclease Activity
Types of Ribonuclease Inhibitors
Preparation of Equipment and Reagents
Other Reagents Used to Control Nuclease Activity
Protocol: Synthesis of Vanadyl Ribonucleoside Complexes
Chapter 5: 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
Chapter 6: The Truth About Tissues
Rationale
Tissue Culture or Tissue?
Homogenization Methods
RNA Isolation Strategies for Various Organs and Tissues
Protocol: LiCl–Urea Method for RNA Isolation from Tissue
Protocol: RNA Isolation from Lipid-Enriched Tissue
Purification of Polysome-Engaged mRNA
Collecting Samples in the Field
RNA “Clean-Up¿ Methods
Chapter 7: 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
Rapid, Non-Column Poly(A)+ Purification
Chapter 8: Quality Control for RNA Preparations
Rationale
Quality Control Technique 1: Electrophoretic Profile of the RNA
Quality Control Technique 2: Ultraviolet Spectrophotometry and Absorption Ratios
Quality Control Technique 3: Sample Capacity to Support RT-PCR
Quality Control Technique 4: Northern Analysis
Quality Control Technique 5: Sample Capacity to Support In Vitro Translation
Chapter 9: Dot Blot Analysis
Rationale
Advantages and Disadvantages
Appropriate Positive and Negative Controls
Limitations of the Data
Chapter 10: Electrophoresis of RNA
Rationale
Normalization of Nucleic Acids
RNA Denaturing Systems for Agarose Gel Electrophoresis
Molecular Weight Standards
Gel Staining Techniques
Safety Considerations in Electrophoresis
Maintenance of Electrophoresis Equipment
Running Agarose Gels for the First Time: A Few Tips
Chapter 11: Photodocumentation and Image Analysis
Rationale
Photodocumentation
Digital Image Analysis
Chapter 12: Northern Analysis
Rationale
Choice of Filter Membrane
Handling and Filter Preparation
Northern Transfer Techniques
Post-Transfer Handling of Filters
Immobilization Techniques
Postfixation Handling of Filters
Chapter 13: Nucleic Acid Probe Technology
Rationale
Probe Classification
Selection of Labeling System
DNA Probes
Antisense RNA Probes
Probe Purification
Probe Storage
Internal Controls
Chapter 14: Practical Nucleic Acid Hybridization
Rationale
Factors Influencing Hybridization Kinetics and Specificity
Hybridization Temperature
Hybridization and the Northern Analysis
Chapter 15: Principles of Detection
Rationale
Autoradiography1
Nonisotopic Procedures
Digital Imaging Systems
Chapter 16: Quantification of Specific mRNAs by Nuclease Protection
Rationale
Basic Approach
Probe Selection
Optimization Suggestions
Potential Difficulties
Protocol: Transcript Quantification by S1 Nuclease Assay
Protocol: Transcript Quantification by RNase Protection Assay
Chapter 17: Analysis of Nuclear RNA
Rationale
Transcription Rate Assays
Protocol: Nuclear Runoff Assay6
Protocol: Nuclear Runoff Assay: Alternative Procedure
Protocol: Nuclease Protection and Pulse Label Transcription Assay
Distinguishing Among the Activities of RNA Polymerases I, II, and III
Extraction of Nuclear RNA for Steady-State Analysis
Protocol: Direct Isolation of Nuclear RNA
Protocol: Preparation of Nuclear RNA from Cells Enriched in Ribonuclease
Chapter 18: cDNA Synthesis
Rationale
cDNA Synthesis—An Overview
Assessing Complementary DNA Synthesis Efficiency
Ligation Considerations
Chapter 19: RT-PCR
Rationale
PCR—An Overview
RT-PCR—General Approach
Laboratory Design
Primer Design
Optimization Procedures
Analysis of PCR Products
RT-PCR Quality Control Points
Related Techniques
Protocol: First-Strand cDNA Synthesis
Protocol: PCR Amplification of cDNA
Cloning PCR Products
Protocol: A-Tailing of Blunt-End PCR Products
Protocol: TA Cloning Ligation Reaction
TOPO Cloning
Chapter 20: Quantitative PCR Techniques
Rationale
Sensitivity Index
Quantitative Approaches
Internal Controls
Exogenous Controls
Control Reaction Formats
Negative Control Considerations
Competitive PCR: Key Considerations
Competitive PCR: Major Steps Involved
Protocol: Competitive PCR
Image Analysis Considerations
Troubleshooting Competitive PCR
Real-Time PCR
Chapter 21: Transcript Subtraction Methods
Rationale
Essential Issues
Subtraction-Suppression PCR
Non-PCR Subtraction
Troubleshooting Subtraction Methods
Chapter 22: mRNA Differential Display
Rationale
General Approach
Product Variety: What to Expect
Protocol: mRNA Differential Display
Protocol: Identification and Selection of Differentially Expressed Sequences
Recovery of Differentially Expressed Sequences by Affinity Capture
Cloning PCR Products
Confirmation of Differential Expression
Subsequent Characterization
Applications of Differential Display
Troubleshooting mRNA Differential Display
Chapter 23: High-Throughput Analysis of Gene Expression
Rationale
What is a Microarray?
What Microarrays Can Do
What Microarrays Cannot Do
Major Steps in Microarray Analysis
Reference RNA
Applications
Chapter 24: RNA Interference: Targeted Gene Silencing
Rationale
Essential RNAi Nomenclature
RNAi—How It Works
siRNA Approach
shRNA Approach
DNA-Directed RNA Interference
Effective Design of siRNAs
Chapter 25: Genomes, Transcriptomes, Proteomes, and Bioinformatics
Rationale
Essential Nomenclature
Genomes and Genomics
Transcriptomes and Transcriptomics
Proteomes and Proteomics
Bioinformatics
Chapter 26: 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: Useful Stock Solutions for the Molecular Biologist
Appendix C: Phenol Preparation
Appendix D: Disposal of Ethidium Bromide and SYBR Green Solutions
Appendix E: DNase I Removal of DNA from an RNA Sample
Appendix F: RNase Incubation to Remove RNA from a DNA Sample
Appendix G: Deionization of Formamide, Formaldehyde, and Glyoxal
Appendix H: Silanizing Centrifuge Tubes and Glassware
Appendix I: Centrifugation as a Mainstream Tool for the Molecular Biologist
Appendix J: Trypsinization Protocol for Anchorage-Dependent Cells
Appendix K: Isolation of High-Molecular-Weight DNA by Salting-Out
Appendix L: RNA Isolation from Plant Tissue
Appendix M: Electrophoresis: Principles, Parameters, and Safety
Appendix N: Polyacrylamide Gel Electrophoresis
Appendix O: Selected Suppliers of Equipment, Reagents, and Services
Appendix P: Useful SI Units
Appendix Q: Common Abbreviations
Appendix R: Trademark Citations
Glossary
Index
