Identification of suitable reference genes for real-time PCR analysis of statin-treated human umbilical vein endothelial cells.

Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-α. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that β-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model

Putative reference genes evaluated.

<p>Putative reference genes evaluated.</p

Details of primers for evaluated genes and RT-qPCR amplification efficiencies.

<p>Details of primers for evaluated genes and RT-qPCR amplification efficiencies.</p

Overall comparison of putative reference genes’ stability for statin-treated cells.

<p>Results are calculated for 10 statin-treated samples for each donor. Rankings are based on <i>geNorm</i> stability M-values, <i>NormFinder</i> stability values and coefficient of correlation values (r) counted by <i>BestKeeper</i>. SD values calculated by <i>BestKeeper</i> are also given in the table. For the overall final ranking the geometric mean of the weights (<i>GeoMean</i>) assigned by the rankings from all three programs was calculated.</p

Overall comparison of putative reference genes’ stability.

<p>Results are calculated for all samples for each donor, i.e. statin-treated cells and combined statin-and-TNF-α-treated cells. Rankings are based on <i>geNorm</i> stability M-values, <i>NormFinder</i> stability values and coefficient of correlation values (r) counted by <i>BestKeeper</i>. SD values calculated by <i>BestKeeper</i> are also given in the table. The genes with SD >1 are eliminated from further analysis. For the overall final ranking the geometric mean of the weights (<i>GeoMean</i>) assigned by the rankings from all three programs was calculated.</p