RNA: Methods and Protocols (Methods in Molecular Biology, 703)

✍ Scribed by Henrik Nielsen (editor)

• Publisher: Humana
• Year: 2010
• Tongue: English
• Leaves: 326
• Category: Library

✦ Synopsis

Recent insight into the transcripts generated from the mammalian genome (i.e. the transcriptome) has revealed that transcription is a far more complex phenomenon than previously thought. In RNA: Methods and Protocols, expert researchers provide the procedures and methods used to describe the structure of messenger RNAs and non-coding RNAs that are transcribed by RNA polymerase II as the immediate gene products in mammalian cells. Focused on the structure of the RNA products of “gene X” and the mapping of proteins associated with these RNAs, the volume presents appropriate information for non-specialists in RNA biology. Written in the highly successful Methods in Molecular Biology™ series format, many chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Comprehensive and practical, RNA: Methods and Protocols views the transcriptional landscape with an appreciation for the role that proteins play in the processing and interpretation of genetic information in an attempt to further our crucial knowledge of the many products and sophisticated regulatory networks that result from it.

✦ Table of Contents

Preface
Organization of the Volume
Part I: RNA Methods
Part II: RNP Methods
Contents
Contributors
Part I Part I RNA Methods
1 The Transcriptional Landscape
1 Introduction
2 The Promoter
3 The Transcription Unit
References
2 Working with RNA
1 Introduction
2 Creating a Working Environment for RNA Work
3 Quantification
4 Extraction with Organic Solvents
5 Desalting and Removal of Nucleotides
6 Gel Purification
7 Precipitation
8 Storage
9 RNA Re-folding
10 Gel Electrophoresis
11 Diethylpyrocarbonate (DEPC) and RNase Inhibitors
12 Notes on a Few Standard Reagents
References
3 Synthesis of RNA by In Vitro Transcription
1 Introduction
2 Materials
2.1 Templates for In Vitro Transcription
2.1.1 Plasmid DNA Templates for In Vitro Transcription
2.1.2 PCR Templates for In Vitro Transcription
2.2 In Vitro Transcription
2.2.1 In Vitro Transcription of Unlabelled Transcripts
2.2.2 In Vitro Transcription of 32P-Labelled Transcripts
2.3 Purification
2.3.1 Purification of Transcripts by Gel Filtration
2.3.2 Gel Purification of Transcripts
3 Methods
3.1 Templates for In Vitro Transcription
3.1.1 Plasmid Templates for In Vitro Transcription
3.1.2 PCR Templates for In Vitro Transcription
3.2 In Vitro Transcription
3.2.1 In Vitro Transcription of Cold (i.e. Unlabelled) Transcripts
3.2.2 In Vitro Transcription of 32P-Labelled Transcripts (see Note 5 for 32P-Handling)
3.3 Purification
3.3.1 Purification of Transcripts by Gel Filtration
3.3.2 Gel Purification of Transcripts
4 Notes
References
4 Efficient Poly(A)+ RNA Selection Using LNA Oligo(T) Capture
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation
3.2 Pre-blocking and LNA-Binding of Streptavidin-Coated Magnetic Particles
3.3 Poly(A)+ RNA Isolation
3.4 Ethanol Precipitation
3.5 Analysis of Purified Poly(A)+ RNA
4 Notes
References
5 Genome Browsers
1 Introduction
2 Getting Started
2.1 UCSC
2.1.1 Genome Browser
2.2 Ensembl
3 Comparative Genomics
3.1 UCSC
3.2 Ensembl
4 Expression and Regulation
4.1 UCSC
4.2 Ensembl
5 Other
5.1 UCSC
5.2 Ensembl
6 Notes
Acknowledgments
6 Web-Based Tools for Studying RNA Structure and Function
1 Introduction
References
7 Northern Blotting Analysis
1 Introduction
2 Materials
2.1 Formaldehyde Agarose Gel
2.2 Glyoxal Agarose Gel
2.3 Denaturing Polyacrylamide Gel
2.4 Hybridization Analysis
3 Methods
3.1 Northern Blotting of a Formaldehyde Agarose Gel
3.2 Northern Blotting of Glyoxalated RNA Separated on an Agarose Gel
3.3 Denaturing Polyacrylamide Gel
3.4 Hybridization Analysis
4 Notes
References
8 Rapid Amplification of cDNA Ends (RACE)
1 Introduction
2 Materials
2.1 Dephosphorylation of Degraded RNA
2.2 Decapping of Intact RNA
2.3 Preparation of RNA Oligonucleotide
2.4 RNA Oligonucleotide--Cellular RNA Ligation
2.5 Reverse Transcription
2.6 PCR Amplifications
2.7 DNA/RNA Purification and Electrophoresis
3 Methods
3.1 Dephosphorylation of Degraded RNA
3.2 Decapping of Intact RNA
3.3 Preparation of RNA Oligonucleotide
3.4 RNA Oligonucleotide--Cellular RNA Ligation
3.5 Reverse Transcription
3.6 First-Round Amplification
3.7 Second-Round Amplification
3.8 Anticipated Results
4 Notes
Acknowledgments
References
9 Fractionation of mRNA Based on the Length of the Poly(A) Tail
1 Introduction
2 Materials
2.1 Molecular Weight Marker
2.2 Template Purification
2.3 Probe Synthesis
2.4 Polyadenylation
2.5 Analysis of Probes
2.6 Oligo(dT)-Based Fractionation of RNA
2.7 Analysis of Fractionation
3 Methods (see Note 1)
3.1 Molecular Weight Marker
3.2 Template Purification by Phenol Extraction and Ethanol Precipitation
3.3 Probe Synthesis by In Vitro Transcription
3.4 Polyadenylation
3.5 Analysis of Probes on a 5% Acrylamide/Urea/TBE Mini Gel
3.6 Oligo(dT)-Based Fractionation of RNA
3.7 Analysis of the Fractionation on a 5% Acrylamide/ UREA/TBE Maxi Gel
3.8 Further Analysis
4 Notes
References
10 Analysis of Mutations that Influence Pre-mRNA Splicing
1 Introduction
1.1 Patterns of Alternative Splicing
1.2 Mechanism of Splice Site Selection
1.3 Single Nucleotide Changes Can Influence Splicing Pattern
1.4 Bioinformatics Resources for Alternative Splicing
1.5 Reporter Gene Analysis of Splicing Events
2 Materials
2.1 Cloning of the Minigenes
2.2 Transfection
2.3 In Vivo Splicing Assay
3 Methods
3.1 Bioinformatics Analysis
3.2 Experimental Testing of the Bioinformatics Prediction
3.2.1 Generation of Vectors with pSpliceExpress
3.2.2 Transfection and Analysis of Minigenes (see Note 3)
4 Notes
Acknowledgment
References
11 S1 Nuclease Analysis of Alternatively Spliced mRNA
1 Introduction
2 Materials
2.1 Labelling of the Oligonucleotide
2.2 S1 Nuclease Assay
2.3 Denaturing Polyacrylamide Gel Electrophoresis
3 Methods
3.1 Design of the Oligonucleotide Probe
3.2 Finding the Optimal Hybridization Temperature
3.3 Finding the Optimal Enzyme and Probe Concentration
3.4 Labelling of Oligonucleotides Using T4 PNK
3.5 S1 Nuclease Assay
4 Notes
References
12 Promises and Challenges in Developing RNAi as a Research Tool and Therapy
1 Introduction
2 RNAi Pathway
3 Design Rules
4 Detection of Exogenous RNAs by Innate Immune Receptors
5 What Is the Nature of IFN-Inducing Motif Present in One Sequence But Absent in Another
6 The Molecular Basis of RNA Sensing by RIG-I
7 Separation of siRNA Unwanted Effects from Gene Silencing
8 Sequence-Dependent Off-Target Effects
References
13 Inhibition of Gene Function in Mammalian Cells Using Short-Hairpin RNA (shRNA)
1 Introduction
2 Materials
2.1 Oligonucleotides and Plasmids Used for Cloning
2.2 Cell Culture and Transfection
2.3 Cellular Protein Isolation and Western Blot Analysis
2.4 Total RNA Isolation and cDNA Synthesis
2.5 Real-Time RT-PCR Analysis
3 Methods
3.1 Selection of Anti-MYCN shRNA Target Sites
3.2 Designing Reverse Primers for Anti-MYCN shRNA Cloning
3.3 Construction of Anti-MYCN shRNA Expressing Plasmids
3.4 Transient Transfection of Anti-MYCN shRNAs into a MYCN-Amplified Neuroblastoma Cell Line
3.5 Total Cellular Protein Isolation
3.6 Western Immunoblot Analysis
3.7 Total RNA Isolation
3.8 cDNA Synthesis
3.9 Real-Time PCR Analysis
3.10 Results
4 Notes
References
14 Validation of RNAi by Real Time PCR
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 RNA Isolation (see Note 2)
2.3 Reverse Transcription
2.4 Real Time PCR
3 Methods
3.1 Tissue Culture
3.2 RNA Isolation
3.3 First Strand cDNA Synthesis
3.4 Real Time PCR
3.4.1 Setting Up the Reaction
3.4.2 Preparation of the Real Time PCR Standard
3.4.3 Analysing the Results
3.4.4 Example of Results
4 Notes
References
15 Profiling RNA Polymerase II Using the Fast ChromatinINTbreak; Immunoprecipitation Method
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers and Solutions
2.3 Equipment
3 Methods
3.1 Cross-Linking
3.1.1 Tissue Culture
3.1.2 Fresh or Frozen Tissue
3.1.3 Yeast Culture
3.2 Lysis
3.3 Sonication
3.4 Isolation of Total DNA (Input Sample)
3.5 Immunoprecipitation
3.6 PCR and Calculation of Enrichment
4 Analysis
5 Notes
Acknowledgment
References
Part II Part II RNP Methods
16 The Post-transcriptional Operon
1 Introduction
2 Coordination of Gene Expression
3 The Post-transcriptional Operon Model of Co-regulated Gene Expression
3.1 Basic Observations in Support of the Importance of Post-transcriptional Regulation and the Post-transcriptional Operon Model
3.2 Examples of Post-transcriptional Operons
4 Conclusions
References
17 RIP-Chip Analysis: RNA-Binding Protein Immuno precipitation-Microarray (Chip) Profiling
1 Introduction
2 Materials
3 Methods
3.1 Preparation of PLB Lysates
3.2 Coating of Bead Matrix with Antibody
3.2.1 Coating Sepharose Beads
3.2.2 Coating Magnetic Beads
3.3 Immunoprecipitation of RNA Binding Protein-mRNA Complex (RIP)
4 Purification of RNA
4.1 Assessment of RNA Quality
4.2 Analysis of Immunoprecipitated RNA
4.3 Synthesis of Labeled cRNA and Microarray Hybridization
4.4 Statistical Analysis of Ribonomic Data from Microarrays
5 Notes
Acknowledgments
References
18 Isolation of RNP Granules
1 Introduction
2 Materials
2.1 Generation of a Cell-Line Expressing 3X FLAG-Tagged RNA-Binding Protein Under Tetracycline Control
2.2 Cell Lysis and Capturing of Granules Containing 3X FLAG-Tagged RNA-Binding Protein
3 Methods
3.1 Generation of a Cell-Line Expressing 3X FLAG-Tagged RNA-Binding Protein Under Tetracycline Control
3.1.1 Construction of a Host-Cell Line
3.1.2 Insertion of the Gene of Interest
3.1.3 Adjustment of Gene Expression by Tetracycline Induction
3.2 Cell Lysis and Capturing of Granules Containing 3X FLAG-Tagged RNA-Binding Protein
4 Notes
Acknowledgements
References
19 Electrophoretic Mobility Shift Assay for Characterizing RNA--ProteinINTbreak; Interaction
1 Introduction
2 Materials
2.1 General Methods
2.2 Preparation of Radiolabeled RNA
2.3 EMSA to Characterize RNA--Protein Interaction
3 Methods
3.1 General Methods
3.1.1 RNase-Free Technique
3.1.2 Phenol/Chloroform Extraction of RNA Solutions (Removal of Protein)
3.1.3 Precipitation of RNA Solutions
3.2 Preparation of Radiolabeled RNA
3.2.1 Dephosphorylation of RNA with Calf Intestinal Phosphatase (CIP)
3.2.2 5-End Labeling with T4 Polynucleotide Kinase (PNK)
3.2.3 Purification of 5-End Labeled RNA
3.3 EMSA to Characterize RNA--Protein Interaction
3.3.1 RNA--Protein Binding Reactions
3.3.2 Resolving RNA--Protein Complexes by Native Page
3.4 Analysis of EMSA Results
3.4.1 Titration of L7Ae onto sR8 RNA
3.4.2 Further Applications of EMSA
4 Notes
References
20 Polysome Analysis and RNA Purification from Sucrose Gradients
1 Introduction
2 Materials
2.1 Yeast Culture and Preparation of Cell Lysate
2.2 Gradient Preparation
2.3 RNA Isolation from Polysomal Profiles
2.4 RNA Electrophoresis
3 Methods
3.1 Yeast Culture and Preparation of Cell Lysate
3.2 Gradient Preparation and Centrifugation (for SW41 Beckman Rotors)
3.3 Data Acquisition and Normalization
3.4 RNA Isolation from Polysome Profiles (for SW41 or SW28 Profiles Split into Two Fractions)
3.5 Electrophoresis of RNA in TAE/Formamide Agarose Gels
4 Notes
References
21 Prediction of Targets for MicroRNAs
1 The Empirical Basis for microRNA Target Prediction
1.1 Biochemical and Structural Evidence
1.2 Statistical Correlation from High-Through-Put Experiments
2 Guidelines for Accessing Target Predictions
3 Precomputed Predictions
3.1 MAMI
3.2 TargetScan.org
3.3 miRanda
3.4 miRbase-Targets
3.5 Microrna.org
3.6 PicTar
4 Prediction Servers
4.1 RNAhybrid
5 Evaluating Target Predictions
References
22 Outsourcing of Experimental Work
1 Introduction
2 Preparation of Samples for External Analysis
2.1 Preparation of dsDNA Samples
2.2 Preparation of RNA for Transcriptome Analysis
2.3 Preparation of RNA for miRNA Profiling
2.4 Preparation of Protein Samples
Reference
Subject Index