Springer Protocols: Abstract: Quantitative (Real-Time) RT-PCR in Cardiovascular Research

Quantitative (Real-Time) RT-PCR in Cardiovascular Research

By: Kevin John Ashton³, John Patrick Headrick³

Abstract

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Quantitative (real-time) PCR (qPCR) represents a highly sensitive, sequence-specific, and reproducible technique for the gel-free detection and quantitation of nucleic acids. Owing to its large dynamic range and throughput, this approach has become the chosen method for rapid quantification of mRNA levels in biological samples. The sensitivity of this method permits the reliable detection of low concentrations of initial template, while delivering a linear range of up to 10 orders of magnitude in copy number. This chapter details the basic methodology behind key components of a qPCR experiment, including sample preparation, fluorescent chemistries, primer/probe design, and data analysis applicable to cardiovascular research.

Affiliation(s): (3) Heart Foundation Research Centre, Griffith University, Southport, Australia

Book Title: Cardiac Gene Expression: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 366 | Pub. Date: Mar-19-2007 | Page Range: 121-143 | DOI: 10.1007/978-1-59745-030-0_7

Subject: Genetics/Genomics

Key Words: Real-time quantitative polymerase chain reaction - reverse transcription - SYBR green I - 5′-nuclease probes - TaqMan - molecular beacons - standard curve - comparative delta C T - primer design

References

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