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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping
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Published on: May 25, 2015

High-throughput real-time quantitative reverse transcription PCR.

Angie L Bookout1, Carolyn L Cummins, David J Mangelsdorf

  • 1Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
Summary
This summary is machine-generated.

This guide details real-time quantitative PCR (QPCR) for gene expression analysis. It covers primer design, relative quantification methods (DeltaCt, DeltaDeltaCt), and absolute RNA quantification for high-throughput applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Gene expression analysis is crucial for understanding biological processes.
  • Real-time quantitative PCR (QPCR) is a powerful technique for quantifying nucleic acids.
  • High-throughput analysis demands robust and adaptable protocols.

Purpose of the Study:

  • To provide comprehensive protocols for gene expression analysis using QPCR.
  • To detail methods for both relative and absolute quantification of RNA.
  • To adapt protocols for high-throughput, 384-well-format instruments.

Main Methods:

  • Detailed protocols for real-time quantitative PCR (QPCR).
  • Primer and probe design and validation strategies.
  • Three relative quantification methods: standard curve, DeltaCt, and DeltaDeltaCt.
  • Absolute quantification using RNA standards and RT-PCR.
  • Production and quantitation of synthetic RNA molecules.

Main Results:

  • Established protocols for high-throughput QPCR gene expression analysis.
  • Validated methods for relative quantification (standard curve, DeltaCt, DeltaDeltaCt).
  • Developed a method for absolute RNA quantification accounting for reaction efficiencies.
  • Demonstrated the production and quantitation of RNA standards for RT-PCR.

Conclusions:

  • The provided protocols enable accurate and efficient gene expression analysis via QPCR.
  • The methods are adaptable for various real-time PCR instruments and high-throughput applications.
  • The study facilitates both relative and absolute quantification of RNA, crucial for molecular research.