title: Identification of suitable reference genes for real-time PCR analysis of statin-treated human umbilical vein endothelial cells.
abstract: 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.
authors.affiliation: Department of General and Nutritional Biochemistry, Medical University of Warsaw, Warsaw, Poland. barbara.zyzynska@wum.edu.pl
authors.forename: Barbara
authors.initials: B
authors.lastname: Żyżyńska-Granica
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Koziak
mesh_terms: D001681:Biological Assay; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D014019:Tissue Donors
chemical_list: D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors