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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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RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization.

Tomas Hruz1, Markus Wyss, Mylene Docquier

  • 1Department of Biology, ETH Zurich, 8092 Zurich, Switzerland.

BMC Genomics
|March 23, 2011
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Summary

Reference gene selection is crucial for accurate quantitative real-time PCR (RT-qPCR). This study found that commonly used reference genes are not universally stable, recommending context-specific gene selection for reliable gene expression analysis.

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Area of Science:

  • Molecular Biology
  • Bioinformatics

Background:

  • Quantitative real-time PCR (RT-qPCR) is a key technique for gene expression analysis.
  • Normalization using endogenous reference genes is standard practice in RT-qPCR.
  • Expression variability of commonly used reference genes across different experimental conditions is a growing concern.

Purpose of the Study:

  • To evaluate the expression stability of reference genes across diverse biological contexts.
  • To identify optimal reference genes for accurate RT-qPCR data normalization.
  • To introduce a tool for selecting context-specific reference genes.

Main Methods:

  • Analysis of transcriptomic data from a large database of normalized microarray experiments (Genevestigator).
  • Assessment of expression stability and variance of genes across various organisms and conditions.
  • Development and validation of a community tool (RefGenes) for reference gene selection.

Main Results:

  • No single reference gene demonstrates universal stability across all biological contexts.
  • Frequently used reference genes often exhibit high transcript abundance and variable expression.
  • Context-specific stable reference genes with lower variance were identified for various biological conditions.

Conclusions:

  • Normalization of RT-qPCR data requires reference genes tailored to specific experimental conditions.
  • The RefGenes tool aids in selecting appropriate reference genes for improved RT-qPCR accuracy.
  • Validation studies confirmed that RefGenes-selected candidates outperform commonly used reference genes.