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Rio, Donald C.; Ares, Manuel Jr.; Nislon, Timothy w.:
RNA: A Laboratory Manual
Preis:
€ 98,00
Einband: Paperback
Auflage: 1. Auflage
Verlag: Cold Spring Harbor Laboratory Press
Erscheinungsdatum: 12/2010
Seiten: 600 pp.
Abbildungen: illus., appendices, index
ISBN-10: 0-87969-891-8
ISBN-13: 9780879698911
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Beschreibung
So much has been learned about RNA in the past 10 years that the ability to purify, analyze, and manipulate RNA molecules is now essential in all kinds of bioscience. Initiating RNA research can be intimidating, but the new book RNA: A Laboratory Manual provides a broad range of up-to-date techniques presented in a functional framework, so that any investigator can confidently handle RNA and carry out meaningful experiments, from the most basic to the highly sophisticated. Originating in three of the field's most prominent laboratories, this manual provides the necessary background and strategies for approaching any RNA investigation, as well as detailed protocols and extensive tips and troubleshooting information. It is required reading for every research laboratory in the life sciences.
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Inhalt
Contents
(preliminary)
Chapter 1 The Fundamentals
Introduction
Common sense in dealing with RNA
pH
Metal ions
Ribonucleases
Read the protocol!
Purification methods and when to use them
Solubilizing and deproteinizing
TRIzol
Guanidine cesium choloride
Column chromatography
SDS/proteinase K/phenol-chloroform
RNA extraction from tissues
Recovering purified RNA: guidelines for precipitation
Resuspending pelleted RNA
Assessing the quantity and quality of isolated RNA after purification
RNA analysis—never run an ugly gel
Removing ribosomal RNAs
Enrichment of mRNA using oligo(dT) cellulose
Enriching for species of RNA other than mRNAs
Miscellaneous (but important) general items
The importance of master mixes
Proper use of micropipettors
The importance of Km—remembering enzyme kinetics
Specific activity—important considerations
Summary
Chapter 2 Purification of RNA from Natural Sources
Overview
SDS Solubilization and Phenol Extraction
Purification of RNA Using TRI-reagent (TRIzol)
Ethanol Precipitation of RNA and the Use of Carriers
Guidelines for the Use of RNA Purification Kits
Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells
Removal of Ribosomal RNA Subunits from Cytoplasmic Extract Prior to Solubilization with SDS and Deproteinization
Isolation of Total RNA from Yeast Cell Cultures
Bacterial RNA Isolation (E. coli)
Removing rRNA from Deproteinized, Phenol-Extracted Total RNA
Enrichment of Poly(A)+ mRNA Using Immobilized Oligo(dT)
Removal of DNA from RNA
Gel Electrophoresis
Determining the Yield and Quality of Purified RNA
Chapter 3 Detection and Sequence Determination of Specific RNAs
Probes and how to make them
RNA and DNA-based probes overview
LNA
Kinasing DNA or RNA and oligonucleotides with -32P-ATP and T4 polynucleotide kinase
DNA probes: random hexamer 32P radiolabeling of DNA fragments as hybridization probes
Nick translation
DNA oligonucleotide radiolabeling by terminal deoxynucleotidyl transferase
Single primer (asymmetric DNA probes)
Northern blotting
Northern blots (formaldehyde RNA gel electrophoresis, gel blot transfer, and hybridization)
Northern blots (formaldehyde)
Northern blots (glyoxal RNA denaturation, gel electrophoresis, gel blot transfer, and hybridization)
Northern blot (gel blot setup)
Northern blot protocol for small RNAs and microRNAs
Primer extension analysis
Primer extension analysis of RNA
Reverse primer extension
Poison primer extension
Direct RNA sequence determination
Direct RNA sequencing by primer extension with dideoxynucleoside triphosphates
Chemical RNA sequencing
Direct RNA sequencing by iodoethanol cleavage at phosphorothioate linkages
Nuclease protection
RNase protection assay
S1 nuclease protection mapping
Specific methods of detecting small RNAs
Ligation-based methods
Gel-shift
Stem-loop PCR
RNase H digestion
Other methods
Identification of RNA by 3' labeling—RNase H
PCR approaches
Reverse transcription-polymerase chain reaction (RT-PCR) primary protocol—oligo(dT) or random hexamer primed
Reverse transcription-polymerase reaction (RT-PCR) alternate protocol—gene-specific primer
Q-PCR
5'-RACE
3'-RACE
Chapter 4 Synthesis and Modification of RNA In Vitro
Overview
In vitro transcription
High yield preparative T7 RNA polymerase in vitro transcription reactions
Synthesis of 32P-labeled RNAs with T7/SP6/T3 RNA polymerase using plasmid DNA or oligonucleotide templates
Introduction of modified nucleotides
Gel purification
Preparing capped RNA
HPLC
Purification of synthetic RNA by reversed-phase chromatography
Purification of synthetic RNA by anion-exchange chromatography
Determining the yield and quality of purified RNA
Manipulating and Labeling Synthesized RNA
Overview
Enzymatic dephosphorylation of RNA or DNA using calf intestine or shrimp alkaline phosphatase
5' End labeling of RNA with -32P ATP and T4 polynucleotide kinase
3' End labeling of RNA with 32P pCp and T4 ligase (RNA ligase 1)
3' End labeling of RNA with yeast with poly(A) polymerase and 3'-deoxyadenosine 5'
3' end labeling of RNA with yeast poly(A) polymerase and 3'-deoxyadenosine 5'-[α-32P]-triphosphate (cordycepin 5-[α-32P]-triphosphate
Biotinylation (postsynthesis or isolation)
Preparation of fluorescent dye-labeled cDNA from RNA for microarray
Site-specific techniques
Overview
Site-specific labeling
Site-specific substitution
Chapter 5 Uses of Synthetic RNAs
Overview
RNA–Protein interactions
Overview
Native polyacrylamide gel electrophoresis binding assay for
RNA–protein complexes
Native gel electrophoresis binding assay for RNA–protein complexes
Filter binding assay for analysis of RNA–protein interactions
Ultraviolet photochemical crosslinking to detect RNA binding proteins
In vitro selection (SELEX) of RNA binding protein binding sites from randomized RNA libraries
Footprinting
Enzymatic (RNase protection) footprinting
Hydroxyl radical footprinting
Chemical modification-interference assay for RNA-binding proteins
Modification interference (NAIM)
Structural mapping—chemical and enzymatic
Nuclease protection/reverse footprinting
Crosslinking
Overview
Chemical crosslinking/UV crosslinking in vivo (psoralen), formaldehyde
Affinity purification
Affinity purification of RNA–protein complexes
Biotin–avidin/streptavidin
MS2-MBP
PP7
GST-MS2
RNA aptamers – streptavidin, sepharose, tobramycin, etc.
Tethering approaches (RNA epitope tagging) and fusion proteins (in vivo and in vitro)
Chapter 6 Testing RNA Substrates for Activity In Vitro
Overview
Extract preparation
Preparation of extracts (overview)
HeLa
Preparation of Drosophila Kc cell nuclear extracts for in vitro splicing
Yeast
In vivo splicing
Splicing reactions (HeLa)
In vitro splicing reactions in Drosophila Kc nuclear extracts
Splicing reactions (yeast)
Chapter 7 RNAi-related Techniques
Drosophila cell culture RNAi
siRNA/mammalian cells
shRNA plasmids and retroviruses
Chapter 8 Genomic Approaches
Overview
Generating RNA
The “right” array (Affy, Agilent, NimbleGen, homemade)
Data analysis/software
HTP Sequencing (454/Solexa/SOLiD)
RIP-ChIP, RIP-seq, CLIP-seq
Appendices
I Reagents
II Stock Solutions
III Nucleases and What They Do
IV Miscellaneous Protocols
V Cautions
VI. Enzymes
Autoreninfo
Donald C. Rio, Department of Molecular and Cell Biology, University of California, Berkeley; Manuel Ares, Jr., Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz; Timothy W. Nilsen, Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, Ohio
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