Oxidative 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). 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, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB. 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 performed by qRT-PCR assays in HUVECs under oxidative stress study.

中文翻译：

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鉴定合适的参考基因，用于实时定量PCR分析过氧化氢处理的人脐静脉内皮细胞。

氧化应激可诱发血管内皮细胞的细胞损伤，这是动脉粥样硬化发展的最初事件。尽管定量实时聚合酶链反应（qRT-PCR）已广泛用于氧化应激损伤的基因表达研究，但使用经过仔细验证的参考基因并未在相关研究中引起足够的重视。因此，本研究的目的是选择一组稳定表达的参考基因，用于在过氧化氢（H2O2）诱导的人脐静脉内皮细胞（HUVEC）氧化应激损伤中的qRT-PCR归一化中使用。使用geNorm分析，我们发现在H2O2处理的HUVEC中，五个稳定表达的参考基因足以用于qRT-PCR分析中的标准化。根据geNorm，表达最稳定的基因是U6，TFRC，RPLP0，GAPDH和ACTB，根据NormFinder，分别是ALAS1，TFRC，U6，GAPDH和ACTB。两者合计，我们的研究表明参考基因的表达稳定性可能会根据所使用的统计程序而有所不同。U6，TFRC，RPLP0，GAPDH和ACTB是在氧化应激研究下通过HURTC中qRT-PCR分析进行基因表达研究的最佳参考基因集。