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Quantification of human PPARgamma1 gene expression by competitive PCR using an homologous internal standard

Nik Soriani Yaacob1, Ruzilawati Abu Bakar, Mohd-Nor Norazmi

  • 1Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. soriani@kb.usm.my

Annals of Clinical and Laboratory Science
|March 25, 2004
PubMed

Insights

A new competitive polymerase chain reaction (PCR) method accurately quantifies gene expression. This technique reliably measures low levels of human peroxisome proliferator-activated receptor gamma 1 (hPPARgamma1) in monocytes.

Area of Science:

  • Molecular Biology
  • Gene Expression Analysis

Background:

  • Standard polymerase chain reaction (PCR) amplifies mRNA but is not ideal for quantifying gene expression due to its exponential nature.
  • Accurate quantification of low-copy-number mRNAs is crucial for understanding cellular processes.

Purpose of the Study:

  • To develop and validate a competitive PCR technique for precise gene expression quantification.
  • To measure the expression levels of human peroxisome proliferator-activated receptor gamma 1 (hPPARgamma1) in activated monocytes.

Main Methods:

  • Construction of an internal standard homologous to, but shorter than, the target gene.
  • Application of competitive PCR to quantify hPPARgamma1 expression in lipopolysaccharide (LPS)-activated monocytes.
  • Validation using a mock system to assess accuracy and sensitivity.

Main Results:

  • The competitive PCR method reliably quantified hPPARgamma1 expression.
  • Accurate quantification was achieved down to the attomole (10^-18) level.
  • Differential expression of hPPARgamma1 was observed in LPS-activated monocytes.

Conclusions:

  • Competitive PCR offers a reliable method for quantifying gene expression, especially for low-abundance targets.
  • The developed method is sensitive and accurate for analyzing gene expression in cellular systems.
  • This technique is valuable for studying the differential expression of genes like hPPARgamma1 during cellular activation.

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