Cellular senescence is recognized as a general response to a variety of oncogenic and genotoxic stresses. It was originally observed in cultures of primary Human Diploid Fibroblasts (HDFs) as they reached the end of their proliferative life span [1]. Normal HDFs enter the senescence state after about 55 - 60 population doublings [2]. Senescent cells have been shown to accumulate with age in human tissues and thus have been proposed to contribute to organismal aging [3]. Senescent cells are resistant to mitogen-induced proliferation, express senescence-associated β-galactosidase (SA β-gal) activity and have an enlarged and flattened morphology [4-7]. Consequently, senescent cells exhibit a gradual loss of replicative potential that results in reduced cell harvest and saturation densities [8].

A distinctive feature of senescent HDFs is that they express elevated levels of p16^INK4a and p21^CIP1 CyclinDependent Kinase (CDK) inhibitors [9-13].

Different roles of p21 and p16 in cellular senescence of human diploid fibroblasts have been reported. Up‐ regulation of p16 might be essential for maintaining cell cycle arrest in cellular senescence, whereas p21 might be responsible for the inactivation of both cyclin E and cyclin D1-associated kinase activity at the early stage of replicative senescence. A series of cyclin-dependent kinases (CDKs) such as CDK2, CDK4 and CDK6 has been re- ported to play a critical role in the phosphorylation of pRb [14,15] which results in pRb loses its ability to bind to E2F/DP transcription factor complexes. As a consequence, cells commit to S-phase of the cell cycle. However, in senescent cells the activity of CDKs is inhibited by CDK inhibitors such as p^21Cip1 and p16^INK4a [10,11,16]. Induction of p16^INK4a and p^21Cip1 prevents phosphorylation of pRb and results in a stable G1 arrest [17]. Thus, CDK inhibitors have the capacity to arrest cells in the G1- phase of the cell cycle and its probable physiological role is in the implementation of irreversible growth arrest termed cellular senescence [18]. In addition, CDK inhibitors specifically p16^INK4a can induce some features of cell senescence [19-23]. Therefore, CDK inhibitor p16 was suggested as an important player in aging and agerelated diseases. Previous study showed that p16 expression was very low or absent in young organisms but was increased with advancing age [24].

Over expression of any of the CDK inhibitors will induce G1 cell cycle arrest. The INK4 (inhibitors of CDK4) family of CDK inhibitors including p15, p16, p18 and p19, bind to and inhibit CDK4 and CDK6. In contrast, the Cip/Kip (CDK2-interacting protein) family of CDK inhibitors (p21, p27 and p57) exhibits a broad specificity for CDKs [25].

CDK inhibitor p16 has emerged as an important player in aging and age-related diseases. p16^INK4a can induce some features of cell senescence [19-23]. p16 expression was very low or absent in young organisms but was increased with advancing age [24].

In recent years, advances in the development of technologies based on short RNAs that silence gene expression and knockdown the production of proteins have been enormous. The initial report on the development of a potent and sequence specific gene silencing by injecting double-stranded RNA (dsRNA) into Caenorhabditis elegans in 1998 sparked the phenomenon known as interference RNA (RNAi) [26,27]. Application of RNAi was restricted to basic research on the mechanisms of gene expression regulation in nematode and flies, until it was found that gene expression in mammalian cells was silenced by transfecting the cells with “short” RNA inhibitors also known as siRNA [28]. Therefore, inhibition of gene expression using the RNAi has become the method of choice for studying gene function in mammalian cells. However, successful knockdown of the target gene requires efficient delivery of short or small interfering RNAs [29].

The efficiency of siRNA delivery can vary between cell types with different delivery methods. Therefore, identifying a suitable housekeeping gene as a positive control for optimizing gene silencing is important to facilitate successful delivery of siRNA without affecting cell viability.

Housekeeping genes or reference genes are essential endogenous regulatory genes that are involved in various processes in the cell such as metabolism, cell structure, gene transcription and homeostasis and are therefore constitutively expressed [30].

The RNA encoding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is universally expressed. GAPDH catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate during glycolysis as well as the reverse reaction in tissues involved in gluconeogenesis. GAPDH has also been implicated in other ubiquitous processes such as DNA replication and repair as well as in apoptosis [31]. In this study, we determined the transfection efficiency for p^16INK4a gene silencing by using GAPDH siRNA as a positive control. The silencing effect of p16^INK4a gene in senescent HDFs was also observed.