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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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Analyzing real-time PCR data by the comparative C(T) method.

Thomas D Schmittgen1, Kenneth J Livak

  • 1Division of Pharmaceutics, College of Pharmacy, Ohio State University, Parks Hall, 500 West 12th Avenue, Columbus, Ohio, OH 43210 USA. schmittgen.2@osu.edu

Nature Protocols
|June 13, 2008
PubMed
Summary
This summary is machine-generated.

Learn about quantitative gene expression analysis. This protocol details the comparative C(T) method (2 (-DeltaDeltaC(T)) method) for relative gene expression studies, offering practical examples.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Quantitative gene expression analysis is crucial for understanding biological processes.
  • Two primary methods exist: absolute quantification and relative quantification.
  • Relative quantification compares gene expression levels to a reference gene.

Purpose of the Study:

  • To provide an overview of the comparative C(T) method for quantitative gene expression.
  • To explain the 2 (-DeltaDeltaC(T)) method for relative gene expression analysis.
  • To present examples of quantitative gene expression data presentation.

Main Methods:

  • The comparative C(T) method (2 (-DeltaDeltaC(T)) method) is detailed.
  • This method normalizes gene expression to an internal control.
  • Standard curves are used for absolute quantification.

Main Results:

  • The protocol outlines the steps for applying the comparative C(T) method.
  • Examples illustrate the practical application of relative gene expression analysis.
  • The 2 (-DeltaDeltaC(T)) method is presented as a key technique.

Conclusions:

  • The comparative C(T) method is a widely used approach for relative gene expression.
  • Understanding this method is essential for accurate gene expression studies.
  • The protocol serves as a guide for researchers in quantitative gene expression.