Validation of EF1α and RPL13a as normalization genes for RT-qPCR analysis of CD31 mRNA expression during the endothelial differentiation of MIAMI cells. The relative fold difference in CD31 mRNA expression levels during the endothelial differentiation of MIAMI cells was determined in order to test the functional use of EF1α and RPL13a as normalization genes for RT-qPCR analysis. Fold change analysis was done using the ΔΔCt method [13, 22] normalizing against EF1α, RPL13a, and GAPDH separately or by averaging the results normalized against both EF1α and RPL13a or EF1α, RPL13a, and GAPDH together. Error bars are shown as standard deviation. All mRNA fold difference and statistical calculations are done relative to Day 0 which is set to the value of 1 and is denoted by the dashed line in each graph. N = 3 independent experiments.