Gene Expression Analysis of RPESCs

Real-time PCR was performed with a Master Cycle (Eppendorf, Hamburg, Germany) apparatus using EVA Green PCR Master Mix (Bio-Rad, Milano, Italy) according to the manufacturer's instructions. Conditions were as follows: denaturation 98 °C for 2 min and 40 cycles of 98 °C for 60 s and 60 °C for 60 s. A melting stage was added at the end of amplification. There was no non-specific amplification as determined by the melting curve. All samples were tested in triplicate with the reference genes β-actin and RPL30 for data normalization. Genes and related primer sequences (SOX2, KLF4, c-MYC, RPE65, CRALBP, PEDF, OTX2, MITF, p21 and p53) were as described previously [58 (link)]. The mRNA expression level of all tested genes was analyzed in young and senescent RPESCs with the 2^-ΔΔCt method: Δ (ΔCt) = ΔCt (senescent) − ΔCt (young). The relative expression values of the genes of interest are reported as mean ± standard deviation (SD) of three independent experiments.