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P5980 Sigma

Gene cloning and analysis by RT-PCR

  •  ISBN-10 1-881299-14-7

  •  ISBN-13 978-1-881299-14-1



Related Categories Books and Software, Labware, Molecular Biology Books, PCR Books
publication info   P. Siebert and J.W. Larrick, ed., Eaton Publishing, 1998, 350 pp., comb bound
mfr. no.   Eaton Publishing


General description

This new laboratory protocols manual provides investigators with up-to-date RT-PCR based methods for gene cloning and analysis. Since RT-PCR was first described for the detection of low-abundance mRNAs, the method has been extended to quantitative measurements of relative and absolute levels of single and multiple mRNAs. Methods for obtaining RNA for RT-PCR have also been extended to include analysis of mRNA levels in clinical samples and even in single cells.

Table of Contents

Section I. Obtaining RNA for RT-PCR
RT-PCR using formalin-fixed, paraffin-embedded (FFPE) archival tumor specimens,
Isolation of RNA from solid tumors,
Isolating single cells for analysis by RT-PCR,
Isolation of RNA from mammalian cells: Application to large mRNA,
RT-PCR without RNA isolation,
Section II. Quantitative RT-PCR
Absolute quantification of messenger RNA using multiplex RT-PCR,
Standard curve quantitative competitive RT-PCR (SC-QC-RT-PCR): A simple method to quantify absolute concentration of mRNA from limited amounts of sample,
Design and use of an exogenously added competitive RNA multiplex template for measurement of mRNA levels by RT-PCR,
Alternative quantitative PCR method,
Quantitation of PCR products using DNA hybridization assays in microplates,
Section III. Cloning Differentially Expressed cDNAs
RNA Display/Fingerprinting: Improvements to differential display
DD/AP-PCR: Differential display & arbitrarily primed PCR of oligo(dT) cDNA
Preferential identification of differentially expressed mRNAs of moderate to low abundance in a microscopic system using selected primers
Solid-phase differential display
cDNA Subtraction: Isolating differentially expressed genes by representational difference analysis
Linker-capture subtraction
Suppression subtractive hybridization: A method for generating subtracted cDNA libraries starting from poly(A+) or total RNA
Optimized subtraction-enhanced display technique
Section IV. Obtaining Full-Length cDNAs and Homologous cDNAs
Enriched full-length cDNA expression library by RecA-mediated affinity capture
Gene-capture PCR by R. Mastrangeli and S. Donini; Improved technique for walking in uncloned genomic DNA
Section V. Ancillary PCR Methods
Generation and use of high-quality cDNA from small amounts of total RNA by SMART(tm) PCR,
Protein production from PCR products
In vitro cloning: A method for PCR amplification of individual unknown DNA fragments suitable for direct sequencing

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