Quantitative PCR of iPSC-derived RPE

RNA was isolated using the QiaShredder and RNeasy mini kits (Qiagen), treated with RNase-Free DNase 1 (Qiagen), and 0.5 µg was reverse transcribed using the Superscript III Reverse Transcriptase kit (Life Technologies). For the quantitative PCR (qPCR) studies, primer sequences were previously reported as follows: endogenous NANOG, SOX2, OCT4, LIN28A and CHM [13 (link)]; PAX6, ZO1, MERTK, TYR, RLBP1, RDH5 and BEST1 [19 (link)]. RNA from wild type iPSCs [30 (link)] or iPSC-derived RPE [19 (link)] was used as a positive control, and from fibroblasts as a negative control. Reactions were performed in triplicate using the LightCycler^® 480 SYBR Green I Master mix on a LightCycler^® 480 II thermal cycler (Roche) and analyzed as described [19 (link)]. Quantification was performed using the ΔΔCt method and expression levels were normalized to GAPDH expression.