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 p16INK4a 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 p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a 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 p16INK4a gene silencing in HDFs aging model.
