Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts.
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 gamma-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 beta-galactosidase (SA beta-gal) activity. However the expression level of GAPDH was consistent in all treatment groups.


CONCLUSIONS
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.
Zainuddin
Azalina
A
Department of Biochemistry, Faculty of Medicine, National University of Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.
Chua
Kien Hui
KH
Abdul Rahim
Norhazira
N
Makpol
Suzana
S
D048430:Cell Shape; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics; D002648:Child; D002839:Chromans / Q000494:pharmacology; D004171:Diploidy; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012015:Reference Standards; D014810:Vitamin E / Q000031:analogs & derivatives / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002839:Chromans; D016877:Oxidants; D012333:RNA, Messenger; D014810:Vitamin E; C013649:plastochromanol 8; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D001616:beta-Galactosidase