Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts |
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| Authors: | Azalina Zainuddin Kien Hui Chua Norhazira Abdul Rahim Suzana Makpol |
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| Institution: | (1) Department of Biochemistry, Faculty of Medicine, National University of Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia;(2) Department of Physiology, Faculty of Medicine, National University of Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia |
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| 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. |
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