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Related Experiment Videos

Guideline to reference gene selection for quantitative real-time PCR.

Aleksandar Radonić1, Stefanie Thulke, Ian M Mackay

  • 1Charité-Campus Charité Mitte, II. Medizinische Klinik mit Schwerpunkt Onkologie und Hämatologie, Humboldt Universität, Berlin, Germany.

Biochemical and Biophysical Research Communications
|January 7, 2004
PubMed
Summary

Choosing the right reference gene is vital for accurate mRNA quantification. This study found RNA polymerase II to be a reliable reference gene across various tissues and experimental conditions, unlike commonly used genes.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Quantitative real-time PCR (qPCR) is a standard method for mRNA quantification.
  • Accurate mRNA transcription comparisons rely on selecting appropriate reference genes.
  • Traditional reference genes like glyceraldehyde 3-phosphate dehydrogenase and beta-actin have shown variability in different tissues and experimental settings, questioning their suitability.

Purpose of the Study:

  • To evaluate the mRNA transcription profiles of 13 potential reference genes.
  • To identify a stable and reliable reference gene for qPCR across diverse biological samples and experimental conditions.

Main Methods:

  • Quantitative real-time PCR was employed to analyze mRNA expression levels.
  • Expression profiles of 13 candidate reference genes were assessed in 16 different human tissues.

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  • Gene expression was also evaluated in CCRF-HSB-2 cells following stimulation with 12-O-tetradecanoylphorbol-13-acetate and ionomycin.

    • Main Results:

    • "Classical" reference genes (e.g., glyceraldehyde 3-phosphate dehydrogenase, beta-actin) demonstrated unsuitable variability.
    • RNA polymerase II exhibited the most consistent mRNA expression across the 16 different tissues analyzed.
    • RNA polymerase II also showed stable expression in CCRF-HSB-2 cells after stimulation with PMA and ionomycin.

    Conclusions:

    • Commonly used reference genes are not universally reliable for qPCR normalization.
    • RNA polymerase II is identified as a highly stable and suitable reference gene for mRNA quantification in various human tissues and stimulated cell lines.
    • The findings provide critical guidance for researchers aiming for accurate and reproducible gene expression studies using qPCR.