Selection of Reference Genes for Normalization of Gene Expression After Exposure of Human Endothelial and Epithelial...

The selection of a stably expressed reference gene is a critical step for the quantitation of gene expression by qRT-PCR. We tested the stability of expression of nine putative reference genes in normoxia and hypoxia in four different human cell types: coronary (HCAECs) and pulmonary endothelial cells (HPAECs), EA.hy926 endothelial cells, and A549 alveolar epithelial cells. Cells were cultured in normoxic and hypoxic conditions for up to 72 h. Total RNA was isolated and used for qRT-PCR. Stability of expression was assessed by calculating the coefficient of variation of the cycle threshold (Ct CV) by pairwise comparison of ΔCt values, and by the NormFinder algorithm. A final rank was calculated for each gene. Finally, we analyzed VEGFA expression by using GAPDH or the optimal candidate reference gene found in this study. Gene expression was variable between cell lines and experimental conditions. The most stable reference gene across all cell lines was TBP, followed by RPLP1 and RPL13A. VEGFA expression was significantly upregulated by 4-fold in hypoxia when using TBP as reference, whilst this result was insignificant when GAPDH was used. The selection of a stably expressed reference gene is a critical step for the generation of reliable and reproducible data in gene expression studies. The most appropriate reference gene may vary in different cell lines and experimental conditions; it should be chosen individually for each experimental set-up.