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Effect of Experimental Treatment on GAPDH mRNA Expression as a Housekeeping Gene in Human Diploid Fibroblasts

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Effect of Experimental Treatment on GAPDH mRNA Expression as a Housekeeping Gene in Human Diploid Fibroblasts

Azalina Zainuddin et al. BMC Mol Biol.

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 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.

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.

Figures

Figure 1
Figure 1
Morphology of human diploid fibroblasts with different experimental treatments. HDFs at young age, passage 4 (A) and senescent (passage 30) (B). The senescent cells showed increased cytoplasm volume and vacuoles, and loss its original fibroblastic shape by acquiring flattened feature. Cells with H2O2-induced oxidative stress showed similar morphological changes like senescent cells (C). The γ-tocotrienol-treated cells showed similar morphology to young cells (D). Micrographs are shown at ×200 magnification.
Figure 2
Figure 2
Expression of senescence associated (SA) β-galactosidase with different experimental treatments. Senescent HDFs, passage 30 (A); cells with H2O2-induced oxidative stress (B); HDFs at young age, passage 4 (C) and γ-tocotrienol-treated HDFs (D). Cells with blue staining indicated positive for β-galactosidase activity. Micrographs are shown at ×200 magnification.
Figure 3
Figure 3
GAPDH mRNA expression. The Ct value showed the expression level of GAPDH was constant (within the acceptable range of 13-16) for the total RNA concentration in the range of 50-100 ng/μl (A). The melting curve demonstrated all PCR products had the specific melting temperature at 87°C (B). Agarose gel electrophoresis confirmed the size of all the PCR products generated was at 217 bp (C). The standard curve showed the threshold cycle (Ct) versus concentration of the total RNA from serial dilution (D).

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