Abstract
BackgroundOxidative stress can 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 received sufficient attention in related studies. 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).ResultsUsing 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, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.ConclusionTaken 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 performed by qRT-PCR assays in HUVECs under oxidative stress study.
Highlights
Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis
This study provides a basis for the selection of reference genes and useful guidelines for future gene expression studies in human umbilical vein endothelial cells HUVECs exposed to hydrogen peroxide (H2O2)
All other genes had median Ct values larger than 20, while pumilio RNA binding family member 1 (PUM1) presented with the lowest expression level with a median Ct value of 31.2. 18S ribosomal RNA (18S) had the widest range of 14.83 cycles, whereas 5′-aminolevulinate synthase 1 (ALAS1) had the narrowest range of 2.665 cycles
Summary
Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. The objective of this study, 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). The initial event in its development is vascular endothelial injury induced by oxidative stress, which is associated with changes in gene expression [2, 3]. Gene expression studies are of great importance to oxidative stress injury research Under pathological conditions, such as ischemia–reperfusion and inflammation, reactive oxygen species (ROS) are generated and lead to vascular endothelial injury [4]. Analysis of gene expression under different physiological and pathological conditions, including oxidative stress, often uses quantitative real-time polymerase chain reaction (qRT-PCR) because of its low template input requirement, high sensitivity, and high specificity [8, 9]. This assumes that expression of the reference gene remains constant in all cell/ tissue types under specific experimental conditions
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