ISBN-10:
0879698918
ISBN-13:
9780879698911
Pub. Date:
12/28/2009
Publisher:
Cold Spring Harbor Laboratory Press
RNA: A Laboratory Manual

RNA: A Laboratory Manual

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Overview

RNA molecules participate in and regulate a vast array of cellular processes, and the scientific community is now entering a new era in which some aspect of RNA biology—as a tool, a therapeutic, a diagnostic, or part of a fundamental process—is becoming increasingly important. But initiating RNA research can be intimidating, and without a thorough understanding of the challenges and complexities inherent in handling this fragile nucleic acid, forays into the RNA world can be quite frustrating. RNA: A Laboratory Manual provides a broad range of up-to-date techniques so that any investigator can confidently handle RNA and carry out meaningful experiments, from the most basic to the most sophisticated. Originating in four of the field's most prominent laboratories and written with novices as well as more advanced researchers in mind, this manual provides the necessary background and strategies for approaching any RNA investigation in addition to detailed step-by-step protocols and extensive tips and troubleshooting information. RNA: A Laboratory Manual will enable any researcher to approach a wide variety of RNA-related problems with confidence and a high expectation of success.

Product Details

ISBN-13: 9780879698911
Publisher: Cold Spring Harbor Laboratory Press
Publication date: 12/28/2009
Edition description: Lab Manual
Pages: 586
Product dimensions: 10.90(w) x 8.40(h) x 1.10(d)

Table of Contents

Preface
Acknowledgments

CHAPTER 1: THE FUNDAMENTALS
Introduction
Common Sense in Dealing with RNA
Purification Methods and When to Use Them
Recovering Purified RNA: Guidelines for Precipitation
Resuspending Pelleted RNA
Assessing the Quantity and Quality of Isolated RNA after Purification
Removing Ribosomal RNAs
Enrichment of mRNA Using Oligo(dT) Cellulose
Enriching for Species of RNA Other Than mRNAs
Miscellaneous (but Important) General Items
Summary

CHAPTER 2: PURIFICATION OF RNA FROM NATURAL SOURCES
Overview
Protocols
1 SDS Solubilization and Phenol Extraction
2 Purification of RNA Using TRIzol (TRI Reagent)
3 Ethanol Precipitation of RNA and the Use of Carriers
4 Guidelines for the Use of RNA Purification Kits
5 Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells
6 Removal of Ribosomal Subunits (and rRNA) from Cytoplasmic Extracts before Solubilization with SDS and Deproteinization
7 Isolation of Total RNA from Yeast Cell Cultures
Method 1: Vegetative Cells
Method 2: Meiotic Cells
8 Bacterial RNA Isolation (E. coli)
9 Removing rRNA from Deproteinized, Phenol-extracted Total RNA
Method 1: Enzymatic Digestion of rRNA
Method 2: Selective Precipitation of Large RNAs with LiCl or PEG/NaCl
Method 3: Removal of rRNA by Hybrid Selection
10 Enrichment of Poly(A)+ mRNA Using Immobilized Oligo(dT)
11 Removal of DNA from RNA
12 Gel Electrophoresis
Method 1: Polyacrylamide Gel Electrophoresis
Method 2: Agarose Gel Electrophoresis (Nondenaturing)
13 Determining the Yield and Quality of Purified RNA

CHAPTER 3: DETECTION AND CHARACTERIZATION OF SPECIFIC RNAS
Overview
Probe Preparation
Northern Blotting
Nuclease Protection
Primer Extension
Other Methods
PCR-based Approaches
Specialized Methods for Detecting Small RNAs
Protocols
1 Labeling of Oligonucleotide Probes (DNA, LNA, RNA) by Polynucleotide Kinase and [?-32P]ATP
2 DNA Oligonucleotide Radiolabeling by Terminal Deoxynucleotidyl Transferase (TdT)
3 Asymmetric Polymerase Chain Reaction to Generate Single-stranded Probes
4 Random Hexamer 32P Radiolabeling of DNA Fragments as Hybridization Probes
5 Nick Translation of Double-stranded DNA for the Preparation of 32P-Labeled Hybridization Probes
6 Enzymatic Dephosphorylation of RNA Using Calf Intestine Alkaline Phosphatase or Shrimp Alkaline Phosphatase
7 Northern Blots: Denaturation and Electrophoresis of RNA with Formaldehyde
8 Northern Blots: Denaturation and Electrophoresis of RNA with Glyoxal
9 Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Hybridization Conditions
10 Northern Blots: Alternative Method for Processing Northern Blots after Capillary Transfer
11 Northern Blots for Small RNAs and MicroRNAs
12 RNase Protection Assay
13 Nuclease S1 Protection Mapping
14 Primer Extension Analysis of RNA
15 Reverse Primer Extension
16 Poisoned Primer Extension
17 Detection of RNAs by 3'-end Labeling and RNase H Digestion
18 Reverse Transcription-Polymerase Chain Reaction
Method 1: Reverse Transcription (RT): cDNA Priming and Synthesis
Method 2: Polymerase Chain Reaction and Detection of Products
19 Basic Quantitative PCR Using Real-time Fluorescence Measurements
20 5' Rapid Amplification of cDNA Ends
21 3' Rapid Amplification of cDNA Ends
22 RACE on Uncapped or Nonpolyadenylated RNAs
23 RNA Sequencing by Primer Extension
24 Direct Chemical Sequencing of End-labeled RNA
25 Splinted Ligation Method to Detect Small RNAs

CHAPTER 4: SYNTHESIS, PURIFICATION LABELING, AND SUBSTITUTION OF TRANSCRIPTS SYNTHESIZED IN VITRO
Overview
In Vitro Transcription: The Basics
Yield of In VitroTranscribed RNAs
Labeling of In VitroSynthesized RNAs
Protocols
1 In Vitro Transcription of RNA: Synthesis, Labeling, and Substitution
2 High-yield Synthesis of RNA Using T7 RNA Polymerase and Plasmid DNA or Oligonucleotide Templates
3 Determining the Yield of RNA Synthesized In Vitro
4 Gel Purification of RNA
5 3'-End Labeling of RNA with [32P]pCp and T4 RNA Ligase 1
6 3'-End Labeling of RNA with Yeast Poly(A) Polymerase and 3'-Deoxyadenosine 5'-[?-32P]Triphosphate (Cordycepin 5'-[?-32P]Triphosphate
7 5'-End Labeling of RNA with [?-32P]ATP and T4 Polynucleotide Kinase
8 Site-specific Labeling and Substitution of RNA

CHAPTER 5: DETECTING AND DEFINING RNA—PROTEIN AND RNA—RNA INTERACTIONS
Introduction
General Methods for Detecting RNAProtein Interactions
RNA Structure Probing
Mapping Sites of RNAProtein Interactions
Summary
Protocols
1 Filter-binding Assay for Analysis of RNAProtein Interactions
2 Native Polyacrylamide-gel-electrophoresis-binding Assay for RNAProtein Complexes: Gel Mobility Shift Assay or Electrophoretic Mobility Shift Assay
3 SELEX to Identify Protein-binding Sites on RNA
4 RNA Structure Determination Using Chemical and Nuclease Digestion Methods
Method 1: RNA Structure Determination Using Chemical Methods
Method 2: RNA Structure Determination Using Nuclease Digestion Methods
5 Mapping Sites of RNAProtein Interactions Using Chemical Methods
6 Mapping Sites of RNAProtein Interactions Using Nuclease-based Methods: RNase Footprinting and Reverse Footprinting
Method 1: RNase Footprinting
Method 2: Reverse Footprinting
7 Modification Interference and Nucleotide Analog Interference Mapping
Method 1: Chemical Modification Interference
Method 2: Nucleotide Analog Interference Mapping
8 Boundary Analysis
9 Ultraviolet Photochemical Cross-linking to Detect RNA-binding Proteins
10 Photocross-linking Using Uridine Analogs
11 Detecting RNARNA Interactions Using Psoralen Derivatives
12 Cross-linking with Formaldehyde
13 Basic Affinity Selection Methods
Method 1: Basic Selection with Beads
Method 2: Selection with a Biotinylated Oligonucleotide
Method 3: Selection of a Ribonucleoprotein Using a Complementary Biotinylated Oligonucleotide

CHAPTER 6: ANALYSIS OF RNA-PROCESSING REACTIONS USING CELL-FREE SYSTEMS
Introduction
Guidelines for the Preparation of Active Cell-free Systems
Guidelines for Testing and Optimizing Extracts
Developing Cell-free Systems from Poorly Studied Organisms
Protocols
1 Preparation of Nuclear Extracts from HeLa Cells
2 Analysis of Pre-mRNA Splicing Using HeLa Cell Nuclear Extracts
3 Preparation of Drosophila Kc Cell Nuclear Extracts for In Vitro Splicing
4 In Vitro Splicing Reactions in Drosophila Kc Nuclear Extracts
5 Preparation and Analysis of Cell-free Splicing Extracts from Saccharomyces cerevisiae
6 Analysis of Splicing In Vitro Using Extracts of Saccharomyces cerevisiae
7 Analysis of Splicing Complexes on Native Gels

CHAPTER 7: RNA INTERFERENCE
Introduction
RNAi: The Basics
The Design of RNAi Experiments: General Considerations and Strategies
RNAi with Long dsRNA
Protocols
1 dsRNA-induced RNAi in Drosophila Cells by Soaking
2 dsRNA-induced RNAi in Drosophila Cells by Transfection
3 dsRNA-induced RNAi in C. elegans by Feeding with dsRNA-expressing E. coli
4 RNAi in C. elegans by Injection of dsRNA
5 RNAi in Cultured Mammalian Cells Using Synthetic siRNAs
6 Construction of Transgenic Drosophila Expressing shRNAs in the miR-1 Backbone
7 Creating an miR30-based shRNA Vector
8 Packaging shRNA Retroviruses and Host Cell Infection
9 Infection of Mammalian Cells with Retroviral shRNAs
10 Creating Transgenic shRNA Mice by Recombinase-mediated Cassette Exchange

CHAPTER 8: GENOMIC APPROACHES
Overview
Microarrays
Sequencing of RNAs by High-throughout Methods
Identifying RNAs Associated with Specific Proteins
Analysis of Genomic Data
Protocols
1 Preparation of Fluorescent-dye-labeled cDNA from RNA for Microarray Hybridization
2 Microarray Slide Hybridization
3 Scanning Microarray Slides
4 Tips on Hybridizing, Washing, and Scanning Affymetrix Microarrays
5 Fragmentation of Whole-transcriptome RNA Using E. coli RNase III
6 Preparation of Small RNA Libraries for High-throughput Sequencing
7 Large-scale Immunopurification of RNP Complexes from Drosophila Nucleoplasmic Extracts for Tiling Microarrays (RNA Immunoprecpitation Microarray; RIP-Chip)
8 CLIP (Cross-linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein
9 Preparation of mRNA-Seq Libraries from Poly(A)+ mRNA for Illumina Transcriptome Highthroughput Sequencing
10 Methods for Processing Microarray Data
11 Methods for Processing High-throughput Sequencing Data

APPENDICES
Common Recipes
Quick Reference for Enzymes Commonly Used in RNA Research
Protocols
Protocols
1 RNA-friendly Plasmid Preparation
2 Preparing Cellular DNA from Nuclei or Whole Cells
3 Expression and Purification of Active Recombinant T7 RNA Polymerase from E. coli
4 Preparing Size Markers for Gel Electrophoresis
5 Toeprinting
6 Cell Fractionation
7 hnRNP-Enriched Nucleoplasmic Extracts from Drosophila or Mammalial Tissue Culture Cells
8 Measuring the Length of Poly(A) Tails
Method 1: Low-resolution Measurement of Poly(A) Tails Using Northern Blotting
Method 2: High-resolution Measurement of Poly(A) Tails Using Northern Blotting
Method 3: PCR-based and Ligation-mediated Approaches for Measuring Poly(A) Tail Length

Cautions
Index

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