Cellular senescence has been most widely studied in fibroblast cells in vitro . In many human cells, cellular senescence is characterized by several molecular and cytological markers, such as a large flat morphology, expression of a senescence-associated β-galactosidase activity (SA β-gal) , and altered gene expression . Oxidative stress is a condition that arises when the production of reactive oxygen species (ROS) overwhelms the cellular antioxidant defences. Human diploid fibroblasts (HDFs) can develop a phenotype resembling senescence in response to oxidative stress. It has been reported that treating cells exogenously with certain hydrogen peroxide (H2O2) concentrations can trigger entry into a senescent-like state which is termed 'stress-induced premature senescence' (SIPS) .
The morphology of SIPS cells was found resembled of senescent cells; with gross enlargement and accumulation of granular cytoplasmic inclusions after 2 weeks exposure to low dose of H2O2 . If oxidative stress or free radicals are at least partly responsible for lifespan and aging, it follows that antioxidant should prolong life and retard aging. Tocotrienol is one subclass of vitamin E that can be found abundantly in palm oil, rice bran oil, barley, corn, oats and wheat . It acts effectively as an antioxidant because its hydrogen atom from the hydroxyl group on its chromanol ring can readily be donated to reduce free radicals, each has its own biological activity .
RNA differential displays  and the serial assessment of gene expression  have been applied to explore senescence-associated genes to gain an insight into the molecular mechanisms underlying senescence . The study of the molecular mechanisms underlying senescence has shed light on central aspects of tumor development and has contributed to the research on organismal aging . Quantification of transcription levels of genes plays a central role in the understanding of gene function . Therefore, quantitative real time RT-PCR has become a popular means to assess mRNA expression level; due to its sensitivity, accuracy and ability to amplify mRNA signal . The method allows detection of amplicon accumulation since it is performed using fluorogenic probes or intercalating dyes such as SYBR Green I, rather than by conventional end-point analysis . However, it is essential to control for error between samples when measuring RNA expression. Therefore, in order to control for experimental variations in the amount of RNA used in each quantitative RT-PCR and batch-to-batch variations in PCR reagents, coincident measurement of so-called 'housekeeping' genes has been used for the normalization of target gene expression data .
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 . Choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. In using the relative quantitative RT-PCR method, the cycle thresholds of the genes of interest were compared to the housekeeping genes to determine relative changes in expression . The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions . If these requirements are not fulfilled then normalization to varying internal references can lead to increased 'noise' or erroneous results . If the chosen housekeeping gene fluctuates randomly between samples, then small differences between genes of interest will be missed. Appropriate validation of internal references is therefore crucial to avoid misinterpretations of study findings .
Several genes have been used as housekeeping genes, including β-actin, β2-microglobulin, cyclooxygenase 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase, porphobilinogen deaminase, and the transferring receptor . The RNA encoding 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 and apoptosis . This gene has been used for real time comparative gene expression studies. However, recent research has demonstrated that the expression level of housekeeping genes may be altered due to differences in experimental treatment . Therefore, it is important to validate the stability and elucidate the changes of the housekeeping genes between different experimental conditions. In order to determine the suitability of GAPDH as reference gene in senescent and antioxidant studies, total RNA from HDFs after different experimental treatments were used to rectify the consistency of GAPDH expression in different reaction conditions of human skin fibroblast cells senescent model.