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Related Experiment Videos

Beta-actin--an unsuitable internal control for RT-PCR.

S Selvey1, E W Thompson, K Matthaei

  • 1Genomics Research Centre, School of Health Science, Griffith University, Gold Coast, Parklands Drive, Southport, QLD 4215, Australia.

Molecular and Cellular Probes
|December 12, 2001
PubMed
Summary

Beta-actin mRNA is regulated by matrigel, making it an unreliable internal control in experiments. Researchers should validate housekeeping genes like 18S rRNA for accurate RT-PCR results.

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Area of Science:

  • Molecular Biology
  • Cell Biology

Background:

  • Beta-actin mRNA is commonly used as a housekeeping gene for internal control in gene expression studies.
  • However, beta-actin expression can be regulated under specific experimental conditions, potentially compromising data accuracy.

Purpose of the Study:

  • To investigate the effect of matrigel treatment on beta-actin mRNA expression.
  • To evaluate the suitability of beta-actin as an internal control in matrigel-treated samples.
  • To identify a reliable housekeeping gene for quantitative RT-PCR in this experimental context.

Main Methods:

  • Differential and competitive reverse transcriptase-polymerase chain reaction (RT-PCR) were employed.
  • Beta-actin mRNA levels were quantified and compared to the target gene, membrane type-1 matrix metalloproteinase (MT1-MMP).

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  • Expression of 18 Svedberg Units (S) rRNA was assessed as a potential control.

    • Main Results:

    • Matrigel treatment caused dose-dependent inhibition of beta-actin mRNA.
    • The target gene, MT1-MMP, showed only moderate inhibition.
    • 18S rRNA exhibited consistent and reproducible expression, unaffected by matrigel treatment.

    Conclusions:

    • Beta-actin is significantly regulated by matrigel and is unsuitable as an internal control in this application.
    • 18S rRNA is a more reliable housekeeping gene due to its stability.
    • Researchers must validate housekeeping genes for specific experimental conditions to ensure accurate quantitative RT-PCR results.