Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

Azalina Zainuddin, Chua Kien Hui, Norhazira A. Rahim, Suzana Makpol

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Background: Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H2O2-induced oxidative stress and γ-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.Results: HDFs with different experimental treatments exhibited diverse cell morphology with different expression of senescence-associated β-galactosidase (SA β-gal) activity. However the expression level of GAPDH was consistent in all treatment groups.Conclusion: The study demonstrated that GAPDH is reliable as reference gene for quantitative gene expression analysis in HDFs. Therefore it can be used as housekeeping gene for quantitative real time RT-PCR technique in human diploid fibroblasts particularly in studying cellular senescence.

Original languageEnglish
Article number59
JournalBMC Molecular Biology
Volume11
DOIs
Publication statusPublished - 14 Aug 2010

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Glyceraldehyde-3-Phosphate Dehydrogenases
Essential Genes
Diploidy
Fibroblasts
Messenger RNA
RNA
Genes
Real-Time Polymerase Chain Reaction
Galactosidases
Tocotrienols
Agar Gel Electrophoresis
Cell Aging
Freezing
Oxidative Stress
Gene Expression

ASJC Scopus subject areas

  • Molecular Biology

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Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts. / Zainuddin, Azalina; Kien Hui, Chua; Rahim, Norhazira A.; Makpol, Suzana.

In: BMC Molecular Biology, Vol. 11, 59, 14.08.2010.

Research output: Contribution to journalArticle

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