Quantitative Gene Expression Analysis

A quantitative gene expression analysis was performed essentially as previously described [22 (link)]. The total RNA was isolated according to Chomczynski and Sacchi [23 (link)] and reverse transcribed using RevertAid^TM reverse transcriptase (Thermo Fisher Scientific, Bleiswijk, Netherlands). A gene expression analysis was performed using real-time SYBR Green technology (Eurogentec, Seraing, Belgium). Primer sequences can be provided on request. Beta-actin (ACTB), peptidylprolyl isomerase A/cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L27 (RPL27), and acidic ribosomal phosphoprotein P0 (36B4) were used as the standard housekeeping genes. The relative gene expression numbers were calculated by subtracting the threshold cycle number (Ct) of the target gene from the average Ct of the housekeeping genes (Ct housekeeping) and raising 2 to the power of this difference.