QPCR was performed on E7.5 embryo cDNA as follows. Briefly, reactions were composed of 5 µl 1.2 µM forward and reverse primer pair, 5 µl diluted cDNA template and 10 µl 2× qPCR mix (see Table S3 for primer sequences). Reactions were heated for 10 min at 94°C, then cycled at 20 sec at 94°C, 20 sec at 60°C, and 30 sec at 72°C. The number of cycles to maintain linearity was determined using the real time analysis software. PCR reactions within the linear range were resolved using native PAGE.
