GAPDH expression as a measurement of transfection efficiency for p^16INK4a gene silencing (siRNA) in senescent human diploid fibroblasts

Suzana Makpol^1*, Azalina Zainuddin¹, Kien Hui Chua²

¹Department of Biochemistry Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
²Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.

DOI: 10.4236/ajmb.2012.24041                

Abstract

Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p^16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16^INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16^INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p^16INK4a gene silencing in HDFs aging model.

Keywords

GAPDH; Transfection Efficiency; p^16INK4a siRNA; HDF Aging model

Conflicts of Interest

The authors declare no conflicts of interest.

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