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Related Experiment Videos

Exogenous reference RNA for normalization of real-time quantitative PCR.

Reginald D Smith1, Benjamin Brown, Pranvera Ikonomi

  • 1Laboratory of Chemical Biology, NIDDK, NIH, Building 10, Room 9N318, 10 Center Drive, MSC 1822, Bethesda, MD 20892-1822, USA. smithrd@helix.nih.gov

Biotechniques
|January 28, 2003
PubMed
Summary

Quantitative PCR (qPCR) data normalization is improved using an exogenous standard, a plant gene fragment (ribulose bisphosphate carboxylase or RuBisCO). This method corrects significant variations in gamma-globin mRNA levels in both K562 and CD34+ cells.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Quantitative PCR (qPCR) is a vital technique for gene expression analysis.
  • Accurate normalization is crucial for reliable qPCR results, often relying on endogenous reference genes.
  • Endogenous controls can vary between experimental conditions, introducing potential inaccuracies.

Purpose of the Study:

  • To evaluate the efficacy of an in vitro transcribed 3' mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for qPCR data normalization.
  • To assess the impact of using RuBisCO mRNA as a reference on the measurement of gamma-globin induction in K562 and primary erythroid CD34+ progenitor cells.

Main Methods:

  • An in vitro transcribed 3' mRNA fragment of the RuBisCO gene was used as an exogenous standard.

Related Experiment Videos

  • K562 cells and primary erythroid CD34+ progenitor cells were treated with sodium butyrate.
  • Gamma-globin mRNA levels were quantified using a TaqMan assay.
  • RuBisCO mRNA levels were quantified using a SYBR Green assay.

    • Main Results:

    • Normalization using the exogenous RuBisCO mRNA standard revealed significant differences in gamma-globin induction.
    • A 95% increase in gamma-globin induction was observed in CD34+ progenitor cells after correction.
    • A 44% increase in gamma-globin induction was observed in K562 cells after correction.

    Conclusions:

    • In vitro transcribed RuBisCO mRNA serves as a robust and sample-independent exogenous standard for qPCR normalization.
    • This method enhances the accuracy of gene expression analysis, particularly in studies involving cellular treatments and differentiation.
    • The use of exogenous standards mitigates variations associated with endogenous reference genes in qPCR assays.