RNA Methodologies

RNA Methodologies

A Laboratory Guide for Isolation and Characterization

3rd Edition - April 1, 2005

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  • Author: Robert E. Farrell, Jr.
  • eBook ISBN: 9780080454764

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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

Product details

  • No. of pages: 688
  • Language: English
  • Copyright: © Academic Press 2005
  • Published: April 1, 2005
  • Imprint: Academic Press
  • eBook ISBN: 9780080454764

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

Bench-Current Scientist, Pennsylvania State University, York, PA, USA

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