Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Background. Oxidative stress could induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not cause sufficient attention in related researches. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2). Methods. HUVECs were treated with different concentrations of H2O2, geNorm and NormFinder software were conducted to evaluate the expression stabilities of 15 candidate reference genes. The optimal number of reference genes needed for qRT-PCR was determined using geNorm. Results. Using geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6 and TFRC, RPLP0, GAPDH and ACTB, and were ALAS1, TFRC, U6, GAPDH, and ACTB according to NormFinder. Discussion. Taken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression with qRT-PCR assays in HUVECs under oxidative stress study.