Identification of a novel real-time PCR reference gene panel for EA.hy926 endothelial cells in different growth States and with DHA treatment

Endothelial dysfunction underlies the pathogenesis of many diseases, including atherosclerosis, diabetes, and kidney failure. The EA.hy926 cell line is one of the most widely used human macrovascular endothelial cell models, and has been shown to enter both dysfunctional- and quiescent-like states. However, there are no validated housekeeping genes (HKGs) for EA.hy926 cells, especially suitable for different growth states and fatty acid treatments. Therefore, we screened transcriptomic data of EA.hy926 cells in their growing and quiescent states and after treatment with or without docosahexaenoic acid (DHA), and identified 18 candidate HKGs. Together with eight other commonly used HKGs and one HKG identified from databases, the stability of the 27 candidate HKGs were analyzed with five algorithms: deltaCt, BestKeeper, geNorm, NormFinder, and RefFinder, using the RT-qPCR results of EA.hy926 cells under 4 different growth conditions. The comprehensive ranking results from RefFinder suggested that the top two most stable genes were CAPZB and FBXO7, whereas the least stable commonly used HKGs were RNA18S and ACTB. The suitability of these genes as HKGs for EA.hy926 cells in different growth states and in response to DHA treatment were validated in relation to the expression of selective endothelial markers. The RT-qPCR validation results confirmed that normalization with CAPZB + FBXO7 provided greater sensitivity than the commonly used HKGs in detecting the expression difference of those endothelial markers under different conditions (growth state × DHA treatment). This novel panel of HKGs is available for future studies of EA.hy926 cell responses to different growth states and/or following DHA treatment.