RNA Methodologies Fourth Edition Laboratory Guide for Isolation and Characterization 1st Edition by Robert E. Farrell ISBN 978-0123747273 0123747279
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ISBN 10: 0123747279
ISBN 13: 978-0123747273
Author: Robert E. Farrell Jr.
This is the fourth edition of the successful laboratory guide which has translated the rich story of riboneucleic acid for over fifteen years. RNA Methodologies 4e presents the latest collection of tested laboratory protocols for the isolation and characterization of eukaryotic and prokaryotic RNA with greater emphasis on transcript profiling, including quantification issues and elucidation of alternative transcription start sites. Collectively the chapters work together providing analysis with clear take-home lessons to assist researchers to understand RNA and to optimize time at the bench. The abundant use of flow charts, tables and graphs are especially helpful in the planning and implementation phases of a project and facilitate learning. 30% new material in this edition includes the addition of RNA isolation protocols including RNA isolation from tissue, expansion of PCR optimization analysis and RNA interference sections, the introduction of a new chapter dealing with the molecular biology of plants, and an expanded glossary.
- 30% new material with the addition of RNA isolation protocols including RNA isolation from tissue, expansion of PCR optimization analysis and RNA interference sections, the introduction of a new chapter dealing with the molecular biology of plants, and an expanded glossary
- 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 useful for all labs
Table of contents:
1 RNA and the Cellular Biochemistry Revisited
Why Study RNA?
What is RNA?
Assembly of Polynucleotides
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
Topology of a Typical mRNA Molecule
5′ Cap
Leader Sequence
Coding Region
Trailer Sequence
Poly(A) Tail
Organelle mRNAs
mRNA Stability, Transport, and Turnover
Bicistronic mRNAs
Prokaryotic mRNAs
mRNA Sequence and Structure Affect Translation
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
Gentle Lysis Buffers
Protocol: Isolation of Cytoplasmic RNA by Hypotonic Lysis
Chaotropic Lysis Buffers
Isolation of RNA with Guanidinium Buffers Guanidinium-Acid-Phenol Extraction Techniques
Protocol: Guanidinium-Acid-Phenol Extraction
Density Gradient Centrifugation
Cesium Chloride
Protocol: Cesium Chloride (CsCl) Gradients
Cesium Trifluoroacetate (CSTFA)
Protocol: Cesium Trifluoroacetate (CsTFA) Gradients
Simultaneous Isolation of RNA and DNA
Protocol: Simultaneous Isolation of RNA and DNA
The Word on Kits
Silica Technology
Isolation of Cytoplasmic RNA on a Silica Column
Affinity Matrices
Other Methods
Protocol: Rapid Isolation of RNA with SDS and Potassium Acetate Reagents
Protocol: Isolation of Prokaryotic RNA
Protocol: Isolation of RNA from Yeast
Short- and Long-Term Storage of Purified RNA
References
3 The Truth about Tissues
Rationale
Tissue Culture or Tissue?
Advantages of Cell Culture
Advantages of Tissue Samples
Homogenization Methods
Polytron Disruption
Dounce Homogenization
BeadBeater™ Technique
RNA Isolation Strategies for Various Organs and Tissues
Fresh Tissue
Frozen Tissue
Fixed Tissue
Protocol: LiCl-Urea Method for RNA Isolation from Tissue
Protocol: RNA Isolation from Lipid-Enriched Samples
Purification of Polysome-Engaged mRNA
Protocol: Isolation of Polysomal mRNA
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
Example 1
Example 2
Selection of Polyadenylated Molecules
Magnetic Bead Technology for Poly(A)+ Purification
Oligo(dT)-Cellulose Column Chromatography
Protocol: Purification of Biophysical Quantities of Poly(A) + RNA
Rapid, Non-Column Poly(A)+ Purification
Protocol: Non-Column Poly(A) Purification
References
6 Quality Control for RNA Preparations
Rationale
Quality Control Technique 1: UV Spectrophotometry and Absorption Ratios
Spectrophotometric Methods
Determination of Nucleic Acid Concentration
Determination of Nucleic Acid Purity
Non-spectrophotometric Methods
Quality Control Technique 2: Electrophoretic Profile of the RNA Protocol
Quality Control Technique 3: UV Shadowing Protocol
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
Specific Inhibitors
Vanadyl Ribonucleoside Complex (VDR; VRC)
RNasin®
Non-specific Inhibitors
Preparation of Equipment and Reagents
Diethyl Pyrocarbonate (DEPC)
Alternative: Sterile Water
Hydrogen Peroxide
NaOH and SDS
Other Reagents Used to Control Nuclease Activity
Guanidine Hydrochloride
Guanidine Thiocyanate
Sodium Dodecyl Sulfate
N-Laurylsarcosine
Phenol:Chloroform:Isoamyl Alcohol
8-Hydroxyquinoline
Cesium Chloride
Cesium Trifluoracetate
Proteinase K
RNAlater®
Protocol: Synthesis of VDR
References
8 Stringency: Conditions that Influence Nucleic Acid Structure
Types of Double-Stranded Molecules
Importance of Controlling Stringency
Effect of Salt on Stringency
Effect of pH on Stringency
Effect of Temperature on Stringency
Effect of Formamide on Stringency
Effect of Urea on Stringency
References
9 Electrophoresis of RNA
Rationale
Normalization of Nucleic Acids
Protocol: Poly(A) Normalization
Sample Preparation
RNA Denaturing Systems for Agarose Gel Electrophoresis
Formaldehyde Denaturation
Protocol: Formaldehyde Denaturing Gels
Urea Denaturation
Protocol: Urea Denaturation
Glyoxal/Dimethyl Sulfoxide Denaturation
Protocol: Glyoxalation and Electrophoresis of RNA
Running RNA on Non-denaturing Gels
Molecular Weight Standards
Proper Use of Size Standards
Ribosomal RNA
Gel Staining Techniques
Ethidium Bromide
SYBR Green
SYBR Gold
SYBR® Safe
GelStar®
Silver Staining
Acridine Orange
Methylene Blue
Safety Considerations and Equipment Maintenance
Running Agarose Gels for the First Time: A Few Tips
Essential Vocabulary
Points to Keep in Mind
References
10 Photodocumentation and Image Analysis
Rationale
Safety First
Digital Image Analysis
Image Formats
Practical Considerations
Digital Image Analysis for Every Budget
Image Analysis Workshops
PhosphorImagers
Traditional Methods of Photodocumentation
Sample Visualization
Filtration
Tips for Optimizing Electrophoretograms
Inherent Limitations of Photographic and X-ray Films
References and Suggested Reading
11 Northern Analysis
Rationale
Choice of Filter Membrane
Nitrocellulose
Nylon
Polyvinylidene Difluoride
Handling and Filter Preparation
Northern Transfer Techniques
Capillary Transfer
TurboBlotter™™
Vacuum Blotting
Electroblotting
Alkaline Blotting
Protocol: RNA Transfer by Passive Capillary Diffusion
Protocol: TurboBlotter Downward Transfer of RNA
Post-Transfer Handling of Filters
Formaldehyde Denaturing Systems
Glyoxal Denaturing Systems
Option 1
Option 2
Immobilization Techniques
Baking
Crosslinking by UV Irradiation
Protocol: UV Crosslinking RNA to Nylon Filters
Post-Fixation Handling of Filters
Reverse Northern Analysis
References
12 Nucleic Acid Probe Technology
Rationale
Probe Classification
Selection of Labeling System
Isotope Labeling
Minimizing Decomposition Problems
Non-Isotopic Labeling
The Ubiquitous Dyes Cy3 and Cy5
Popular Chemiluminescence Formats
Biotin
Digoxigenin
Fluorescein
Direct Enzyme Labeling
DNA Probes
DNA Probe Synthesis
Polymerase Chain Reaction
Random Priming
Nick Translation
5′ End-Labeling
3′ End-Labeling
Antisense RNA Probes
Characteristics of RNA Probes
RNA Probe Synthesis
In Vitro transcription
5′ End-Labeling
3′ End-Labeling
Probe Purification
Probe Storage
References
13 Practical Nucleic Acid Hybridization
Rationale
Factors Influencing Hybridization Kinetics and Duplex Stability
Temperature
Ionic Strength
pH
Probe Length
Probe Concentration
G+C Content
Mismatching
Probe Complexity
Viscosity
Formamide
Urea
Hybridization Temperature
Tm for Long Probes
Tm for Oligonucleotide Probes
Hybridization and the Northern Analysis
Prehybridization: Filter Preparation
Protocol: Prehybridization for Long Probes
Probe Denaturation
Hybridization
Posthybridization Stringency Washes
Probe Removal and Rehybridization
Protocol: Generic Method for Probe Removal
References
14 Principles of Detection
Rationale
Autoradiography
Handling of Filter Membranes
X-ray Film
Safe light
Exposure Time
Intensifying Screens
Fluorography
Preflashing Film
Type of Cassette
Development and Fixation
Digoxigenin
Fluorescence Imaging
Direct Enzyme Labeling
Detection by Chemiluminescence
Substract for Chemiluminescence
Chromogenic Detection 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
Relationship to the Study of Steady-State RNA
Nuclear Run-off vs. Nuclear Run-on Assay
Protocol: Nuclear Run-off Assay
Harvesting of Cells and Preparation of Nuclei
Alternative Protocol for Preparation of Fragile Nuclei
Alternative Protocol for Preparation of Nuclei from Whole Tissue
Labeling and Recovery of Transcripts
Preparation of Target DNA
Preparation of RNA for Hybridization
Posthybridization Washes and Detection
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
Protocol: Preparation of Nuclear RNA from Cells Enriched in Ribonuclease
Troubleshoot Nuclear RNA Analysis
References
17 cDNA Synthesis
Rationale
cDNA Synthesis – An Overview
First-Strand Considerations
Reverse Transcriptase Options
Second-Strand Considerations
Classical Methods
PCR-Based Methods
Protocol: First-Strand cDNA Synthesis
Assessing cDNA Synthesis Efficiency
Cloning cDNA
Ligation Considerations
Enzymes Used for Ligation
Applications
References
18 RT-PCR: A Science and an Art Form
Rationale
PCR – An Overview
RT-PCR – General Approach
PCR Carryover Prevention
Laboratory Design
Procedural Methods
Aerosol-Resistant Tips
Uracil-N-Glycosylase
Primer Design
Basic Rules
Tm Considerations
Estimating Tm
Precision Tm Calculations
On-line Resources
Multiplex Primer Design
Optimization Procedures
Thermostable Polymerases
Positive Controls
Negative Controls
Hot Start PCR
Locked Nucleic Acids
Touchdown PCR
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
5′ RACE PCR
5′ RLM-RACE
3′ RACE PCR
Nested PCR
Long-Range PCR
Single Cell PCR
Splinkerette PCR
The Hunt for Alternative Transcription Start Sites
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
Other Amplification Procedures
Linear RNA Amplification (Eberwine Process)
Strand Displacement Amplification
Nucleic Acid Sequence Based Amplification (NASBA)
Ligase Chain Reaction
References
19 Quantitative PCR Techniques
Rationale
Sensitivity Index
Quantitative Approaches
Real-Time PCR
Real-Time PCR Platforms
SYBR Green Assay
TaqMan® Assay
Molecular Beacons
LUXT™ Primers
Scorpions
Melting Curve Analysis
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
Protocol: Competitive PCR
Synthesis of Non-homologous Competitor
Synthesis of First-Strand cDNA
Competitive PCR (Primary Amplification)
Competitive PCR (Secondary Amplification)
Contents
Image Analysis Considerations
Troubleshooting Quantitative PCR Techniques
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
Subtractive Method
Suppression Subtractive Hybridization (SSH)
Troubleshooting
Non-Subtractive Methods
mRNA Differential Display
Protocols
References
23 RNAi: Take a RISC-Role the Dicer
Rationale
Essential RNAi Nomenclature
RNA Interference – How it Works
siRNA Approach
shRNA Approach
siRNA Delivery Methods into Mammalian Cells
miRNA
Effective Design of siRNAs
RNAi and Alternative Transcript Splicing
In Vitro and In Vivo Issues
RNAi Validation
RT-PCR Approaches
Northern Analysis
Western Analysis
Applications
References
24 Genomes, Transcriptomes, Proteomes, and Bioinformatics
Rationale
Essential Nomenclature
Genomes and Genomics
Transcriptomes and Transcriptomics
Protcomes 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
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