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 [γ-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 Vitro–Transcribed RNAs

Labeling of In Vitro–Synthesized 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´-[α-³²P]Triphosphate (Cordycepin 5´-[α-³²P]Triphosphate

7 5´-End Labeling of RNA with [γ-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 RNA–Protein Interactions

RNA Structure Probing

Mapping Sites of RNA–Protein Interactions

Summary

Protocols

1 Filter-binding Assay for Analysis of RNA–Protein Interactions

2 Native Polyacrylamide-gel-electrophoresis-binding Assay for RNA–Protein 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 RNA–Protein Interactions Using Chemical Methods

6 Mapping Sites of RNA–Protein 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 RNA–RNA 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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