Quantitative PCR

RNA samples were extracted using the RNeasy kit and treated with DNase 1 to eliminate DNA contamination (All from Qiagen). cDNA synthesis was performed using the High capacity cDNA reverse transcription kit (ABI). Taqman assay was performed using the Taqman fast advanced master mix (ABI) in triplicates following manufacturer’s protocol, using probes for RAX (Hs00429459_m1), MITF (Hs01117294_m1), ATOH7 (Hs00376955_s1), PAX6 (Hs00240871_m1), BRN3B (Hs00231820_m1), ISL1 (Hs00158126_m1), OPN4 (Hs00264482_m1) and the housekeeping genes β-ACTIN (Hs99999903_m1) or 18S (Hs99999901_s1). Samples were processed using the Step One plus real time PCR system (ABI). The Ct threshold was set using the parameters by the Stepone software (ABI) and checked manually. The results were normalised to the housekeeping genes β-actin or 18S and analysed using the ΔΔCt method. Results were presented in log2 fold changes compared to undifferentiated hESCs and statistical tests were performed using one-way ANOVA test with Dunnett post-hoc test.