RNA Methodologies - 3rd Edition - ISBN: 9780122496967, 9780080454764

RNA Methodologies

3rd Edition

A Laboratory Guide for Isolation and Characterization

Authors: Robert E. Farrell, Jr.
Hardcover ISBN: 9780122496967
eBook ISBN: 9780080454764
Imprint: Academic Press
Published Date: 1st April 2005
Page Count: 688
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Description

This laboratory guide represents a growing collection of tried, tested and optimized laboratory protocols for the isolation and characterization of eukaryotic RNA, with lesser emphasis on the characterization of prokaryotic transcripts. Collectively the chapters work together to embellish the RNA story, each presenting clear take-home lessons, liberally incorporating flow charts, tables and graphs to facilitate learning and assist in the planning and implementation phases of a project.

RNA Methodologies, 3rd edition includes approximately 30% new material, including chapters on the more recent technologies of RNA interference including: RNAi; Microarrays; Bioinformatics. It also includes new sections on: new and improved RT-PCR techniques; innovative 5’ and 3’ RACE techniques; subtractive PCR methods; methods for improving cDNA synthesis.

Key Features

  • Author is a well-recognized expert in the field of RNA experimentation and founded Exon-Intron, a well-known biotechnology educational workshop center
  • Includes classic and contemporary techniques
  • Incorporates flow charts, tables, and graphs to facilitate learning and assist in the planning phases of projects

Readership

Researchers, graduate students and lab technicians in the fields of molecular biology, cell biology, and biochemical genetics.

Table of Contents

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

Details

No. of pages:
688
Language:
English
Copyright:
© Academic Press 2005
Published:
Imprint:
Academic Press
eBook ISBN:
9780080454764
Hardcover ISBN:
9780122496967

About the Author

Robert E. Farrell, Jr.

Dr. Robert Farrell is a bench-current scientist who has 35 years of experience working with RNA in the study of transcriptional and posttranscriptional regulation of gene expression in a variety of model systems. He is also experienced in animal cell culture methods. Prior to joining the faculty at Penn State University, he operated a biotech education and service firm, winning the 1998 Small Business Contractor of the Year award from the U.S. Department of Agriculture. He is the recipient of campus- and college-wide awards for excellence in teaching, and has extensive experience running RNA and specialized biotechnology hands-on laboratory training programs all over the world. He often serves as a consultant within the pharmaceutical and biotech industries. Dr. Farrell received his Ph.D. and M.S. degrees from The Catholic University of America and his B.S. in Biology from Providence College. Dr. Farrell currently serves as the campus academic officer at Penn State York.

Affiliations and Expertise

Penn State University, York, PA, USA

Reviews

Praise for the third edition @qu:"This third edition of RNA Methodologies is an updated version of a laboratory guide edited for the first time in 1993; it represents an exhaustive collection of tried, tested, and optimized protocols for isolation and chracterization of RNA. The third edition of RNA Methodologies is a well-documented guide for any researcher who has to manage RNA studies. Each chapter of this book provides a rationale to help in the decision-making process...The inclusion of troubleshooting hints and annotations based on personal experience are provided to improve the method(s). This laboratory guide offers protocols on traditional and new technologies, with a focus on how to use the expedient technique for achieving an experimental goal in the study of gene expression." @source:--Dr. Patrick Pagesy at INSERM for HEMOGLOBIN