RNA Methodologies

RNA Methodologies: A Laboratory Guide for Isolation and Characterization, Sixth Edition provides the most up-to-date ribonucleic acid lab techniques for seasoned scientists and graduate students alike. This edition features new material on RNA sequencing, RNA in Situ Hybridization, non-coding RNAs, computational RNA biology, transcriptomes and bioinformatics, along with the latest advances in methods and protocols across the field of RNA investigation. As a leader in the field, Dr. Farrell provides a wealth of knowledge on the topic of RNA biology while also giving readers helpful hints and troubleshooting techniques from his own personal experience in this subject area.

This book presents the essential knowledge and techniques to use when working with RNA for the experienced practitioner, while also aiding the beginner in fully understanding this important branch of molecular biology.

Cover image
Title page
Table of Contents
Copyright
Dedication
Preface
Chapter 1. RNA and the cellular biochemistry revisited
Abstract
Why study ribonucleic acid?
What is ribonucleic acid?
Polynucleotide synthesis
Types of ribonucleic acid
Transcription and the central dogma
Promoters, transcription factors, and regulatory elements
Gene and genome organization affect transcription
Ribonucleic acid polymerases and the products of transcription
Hallmarks of a typical messenger ribonucleic acid
Messenger ribonucleic acid—stability and turnover
Bicistronic messenger ribonucleic acids
Prokaryotic messenger ribonucleic acids
Messenger ribonucleic acid sequence and structure affect translation
Levels of gene regulation
References
Further reading
Chapter 2. Creating a ribonuclease-free environment
Abstract
Rationale
Elimination of resilient ribonucleases
Latent RNase contamination issues
Types of ribonuclease inhibitors
Preparation of equipment and reagents
Other compounds used to control nuclease activity
Protocol: synthesis of vanadyl ribonucleoside complexes
References
Further reading
Chapter 3. Stringency: conditions that influence nucleic acid structure
Abstract
Rationale
Types of double-stranded molecules
Importance of controlling stringency
References
Further reading
Chapter 4. RNA isolation strategies
Abstract
Rationale
Goals in the purification of ribonucleic acid
The word on kits
Lysis buffer formulations
Isolation of RNA with guanidinium buffers
Guanidinium–acid–phenol extraction techniques
Density gradient centrifugation
Isolation of RNA and DNA from the same source
Other methods
RNA isolation from fluid matrices
Short- and long-term storage of purified RNA
References
Further reading
Chapter 5. Isolation of polyadenylated RNA
Abstract
Rationale
Polyadenylation
Selection of polyadenylated molecules: how it works
The poly(A) caveat
Magnetic bead technology for poly(A)+ purification
Oligo(dT) affinity chromatography
References
Further reading
Chapter 6. The truth about tissues
Abstract
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
Further reading
Chapter 7. Going green: RNA and the molecular biology of plants
Abstract
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
Protocol: RNA isolation from plant tissue with hot borate
Strategies for RNA isolation from other plant tissues
Troubleshooting RNA isolation from plant tissue
References
Further reading
Chapter 8. Quality control for RNA preparations
Abstract
Rationale
Quality control technique 1: ultraviolet spectrophotometry and absorption ratios
Quality control technique 2: electrophoretic profiling of RNA
Quality control technique 3: RNA integrity number
Quality control technique 4: ultraviolet shadowing
Quality control technique 5: sample capacity to support RT-PCR
Quality control technique 6: sample capacity to support in vitro translation
References
Further reading
Chapter 9. cDNA: a permanent biochemical record of the cell
Abstract
Rationale
cDNA synthesis—an overview
Protocol: first-strand cDNA synthesis
Assessing cDNA synthesis efficiency
Cloning cDNA
Applications
References
Further reading
Chapter 10. RT-PCR: a science and an art form
Abstract
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
Related techniques
The hunt for alternative transcription start sites
Protocol: first-strand cDNA synthesis
Protocol: PCR amplification of cDNA
Cloning PCR products
Other amplification procedures
References
Further reading
Chapter 11. Quantitative PCR techniques
Abstract
Rationale
Sensitivity index
Quantitative approaches
The MIQE guidelines
Real-time PCR
Real-time PCR platforms
Melting curve analysis
Digital PCR
Internal controls
Exogenous controls
Control reaction formats
Negative control considerations
PCR arrays
Competitive PCR: key considerations
Competitive PCR: major steps involved
Alternative approach: nonreal-time competitive PCR
Troubleshooting quantitative PCR techniques
References
Further reading
Chapter 12. miRNA and other noncoding RNAs
Abstract
Rationale
Overview of noncoding RNAs
miRNA structural and functional characteristics
miRNA biogenesis
miRNA profiling
miRNA as key regulator of gene expression
References
Further reading
Chapter 13. RNA interference and gene editing
Abstract
Rationale
Essential RNAi terminology
RNA interference—how it works
Effective design of siRNAs
RNAi and alternative transcript splicing
In vitro and in vivo issues
RNAi validation
RNAi applications
CRISPR-Cas9 and gene editing
References
Further reading
Chapter 14. Electrophoresis of RNA
Abstract
Rationale
Normalization of samples by nucleic acid concentration
Direct measurement of poly(A) content
Intramolecular base-pairing mandates RNA denaturation
Formaldehyde denaturation
Urea denaturation
Glyoxal/dimethyl sulfoxide denaturation
Running RNA on nondenaturing gels
Gel staining options
Safety considerations and equipment maintenance
A few tips for running agarose gels for the first time
References
Further reading
Chapter 15. Photodocumentation and image analysis
Abstract
Rationale
Safety first
Digital image analysis
Traditional methods of photodocumentation
Tips for optimizing electrophoretograms
Further reading
Chapter 16. Northern analysis
Abstract
Rationale
Choice of blotting membrane
Handling and membrane preparation
Northern transfer techniques
Immobilization techniques
Postfixation handling of blotting membranes
Reverse Northern analysis
References
Further reading
Chapter 17. Nucleic acid probe technology
Abstract
Rationale
Factors influencing hybridization kinetics and duplex stability
DNA probe synthesis
Sense and antisense RNA probe synthesis
Selection of labeling system
Mixed phase hybridization: Northern and Southern blots
Principles of detection
Protocol: generic method for probe removal
References
Further reading
Chapter 18. Quantification of specific mRNAs by nuclease protection
Abstract
Rationale
Basic approach
Probe selection
Optimization suggestions
Potential difficulties
Protocol: transcript quantification by S1 analysis
Protocol: transcript quantification by RNase protection
Troubleshooting
References
Further reading
Chapter 19. Analysis of nuclear RNA
Abstract
Rationale
Transcription rate assays
Protocol: nuclear run-on assay
Protocol: alternative procedure for nuclear run-on 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
Protocol: preparation of nuclear RNA from cells enriched in ribonuclease
Troubleshooting nuclear RNA analysis
References
Further reading
Chapter 20. RNA in situ hybridization
Abstracts
Rationale
Technical considerations
Sample preparation
Hybridization and detection procedures
Positive and negative control considerations
Protocol: preparation of fresh frozen brain tissue for in situ hybridization
Protocol: RNA in situ hybridization for zebrafish embryos
Protocol: RNA in situ hybridization whole mount for arachnid embryos
Spatial transcriptomics
In situ hybridization tips for success
References
Further reading
Chapter 21. Array analysis of gene expression
Abstract
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
Further reading
Chapter 22. Subtractive and nonsubtractive methods for the analysis of gene expression
Abstract
Rationale
Essential issues
Nonsubtractive methods
Troubleshooting
References
Further reading
Chapter 23. Transcriptomes and bioinformatics
Abstract
Rationale
Essential vocabulary
Transcriptomes and transcriptomics
The epitranscriptome (epigenetics of RNA)
RNA–chromatin, RNA–RNA, and RNA–protein interactions
Aptamer biology
Bioinformatics
Search for genes—have a BLAST!
References
Further reading
Chapter 24. RNA-seq: the premier transcriptomics tool
Abstracts
Rationale
Essential vocabulary
Overview of RNA-seq
RNA-seq workflow
RNA-seq variations
References
Further reading
Chapter 25. RNA biomarker discovery and validation
Abstract
Rationale
Biomarkers defined
Characteristics of useful biomarkers
miRNA biomarkers
Circulating RNA
Identification of biomarkers for research and diagnostic applications
Biomarker issues and shortcomings
References
Further reading
Chapter 26. Functional genomics strategies
Abstract
Rationale
Functional genomics defined
Importance of functional genomics approaches
Commonly used functional genomics approaches
Relationship of functional genomics approaches to classical molecular biology
References
Further reading
Chapter 27. A few RNA success stories
Abstract
Nucleic acids as pharmaceuticals
A typical experiment?
Sensitivity issues
What to do next
Where to turn for help
References
Further reading
Epilogue
A few pearls of wisdom
Appendix A. Maintaining complete and accurate records
Appendix B. Converting mass to moles
Scenario 1
Scenario 2
Scenario 3
Scenario 4
Appendix C. Removal of DNA from an RNA sample
Protocol: digestion of DNA
Appendix D. Removal of RNA from a DNA sample
Protocol: removal of DNase activity from homemade RNase stock solutions
Protocol: digestion of RNA
Appendix E. Electrophoresis: principles, parameters, and safety
Theoretical considerations
Agarose gel electrophoresis
Polyacrylamide gel electrophoresis
Molecular size range of sample
Nucleic acid conformation
Applied voltage
Ethidium bromide
SYBR dye family
Base composition and temperature
Field direction
Types of gel boxes
Safety considerations in electrophoresis
Maintenance of electrophoresis equipment
Appendix F. Disposal of ethidium bromide and SYBR Green solutions
Protocol 1
Protocol 2
Protocol 3
Protocol 4
Appendix G. Deionization of formamide, formaldehyde, and glyoxal
Appendix H. Silanizing centrifuge tubes and glassware
Protocol
Appendix I. Centrifugation as a mainstream tool for the molecular biologist
Types of centrifuges
Rotors
Applications
Appendix J. Dot blot analysis
Advantages and disadvantages
Appropriate positive and negative controls
Limitations of the data
Protocol: RNA dot blots
Protocol: DNA dot blots
Further reading
Appendix K. Useful stock solutions for the molecular biologist
NOTES
Appendix L. Genomes and proteomes
Genomes and genomics
Proteomes and proteomics
Further reading
Appendix M. Common SI prefixes
Appendix N. Common abbreviations
Appendix O. Select suppliers of equipment, reagents, and services
Appendix P. Trademark citations
Glossary
Index

Life Sciences

Physical Sciences & Engineering

Social Sciences & Humanities

Health

RNA Methodologies

A Laboratory Guide for Isolation and Characterization

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Robert E. Farrell Jr.

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