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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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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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Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR
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Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments.

Stefanie Klenke1, Kristina Renckhoff2, Andrea Engler2

  • 1Klinik für Anästhesiologie & Intensivmedizin, Universität Duisburg-Essen and Universitätsklinikum Essen, Hufelandstr. 55, D-45122, Essen, Germany. Stefanie.Klenke@uk-essen.de.

Naunyn-Schmiedeberg'S Archives of Pharmacology
|September 22, 2016
PubMed
Summary

Accurate mRNA expression analysis using real-time PCR requires careful reference gene selection. This study found poor selection practices and offers an easy tool to improve data reliability for gene expression studies.

Keywords:
H9c2 cellsHypoxiaIsoflurane, preconditioningQuantitative real-time PCRReference gene

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Area of Science:

  • Molecular Biology
  • Pharmacology
  • Biotechnology

Background:

  • Quantitative real-time PCR (qRT-PCR) is crucial for mRNA expression analysis.
  • Reliable qRT-PCR results depend heavily on the appropriate selection of reference genes.
  • Reference gene selection is frequently overlooked in target gene expression studies.

Purpose of the Study:

  • To evaluate the frequency of appropriate reference gene selection in published qRT-PCR studies.
  • To introduce a user-friendly tool for selecting reference genes with minimal variability.
  • To demonstrate the impact of reference gene selection on target gene expression analysis.

Main Methods:

  • Screened 2015 publications in Naunyn-Schmiedeberg's Archives of Pharmacology for qRT-PCR and reference gene selection methods.
  • Analyzed target gene expression (Vegfa, Grk2, Sirt4, Timp3) in H9c2 cells under various conditions (hypoxia, hyperglycemia, isoflurane).
  • Compared target gene expression using the least variability reference gene selection method versus an arbitrarily chosen reference gene (Actb, Gapdh, B2m, Sdha, Rplp1).

Main Results:

  • A significant majority of studies (18 of 21) lacked information on appropriate reference gene selection.
  • Only 1 of 21 papers described a method for reference gene selection; 2 were unclear.
  • Reference gene selection method critically influenced target gene expression interpretation, as shown with Vegfa under hypoxia.

Conclusions:

  • The frequency of appropriate reference gene selection in published qRT-PCR studies is alarmingly low.
  • An easy-to-use strategy for selecting least variable reference genes is proposed.
  • Implementing this strategy can enhance the accuracy and reliability of target gene expression data.