High-throughput real-time quantitative reverse transcription PCR.

Angie L. Bookout, Carolyn L. Cummins, David J. Mangelsdorf, Jean M. Pesola, Martha F. Kramer

Research output: Contribution to journalArticle

170 Citations (Scopus)

Abstract

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Original languageEnglish (US)
JournalCurrent protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
VolumeChapter 15
StatePublished - Feb 2006

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Reverse Transcription
Polymerase Chain Reaction
RNA
Real-Time Polymerase Chain Reaction
Gene Expression

ASJC Scopus subject areas

  • Medicine(all)

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High-throughput real-time quantitative reverse transcription PCR. / Bookout, Angie L.; Cummins, Carolyn L.; Mangelsdorf, David J.; Pesola, Jean M.; Kramer, Martha F.

In: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.], Vol. Chapter 15, 02.2006.

Research output: Contribution to journalArticle

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