Quantifying Neural Cell Lineage Markers

Total RNA was extracted by Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary DNA was synthesized according to the manufacturer’ instructions for RevertAid Reverse Transcriptase reverse transcriptase kit (Thermo Fisher, Waltham, MA, USA). Real-time PCR of neural cell phenotype markers was performed in a Roche 480 Light-Cycler using Light Cycler^®480 DNA SYBR Green I Master (Roche, Basel, Switzerland). Primers and annealing conditions are listed in Table 1. GAPDH was used as a reference gene [24 (link),25 (link),26 (link)]. Data are presented as the differences between reference and samples treated with PPy extracts after normalization to the reference gene by the 2^{−(Cq(target)−Cq(reference))} method [27 (link),28 (link)]. All tests were performed in four separate sets. Data are reported as the mean and standard deviations from a minimum of four independent experiments. Statistical significance was determined by ANOVA with post hoc Tukey’s Multiple Comparison test; * p < 0.05. (*) and (#) mark statistically significant differences compared to ESCs and to EBs without PPy treatment, respectively.