Gene expression analysis by RT–quantitative PCR

Total RNA was prepared with the RNeasy Kit (Qiagen), and 500 ng was reverse‐transcribed using SuperScriptIII (Invitrogen) and oligo‐dT primers according to the manufacturer's protocol. Real‐time PCR was performed using TaqMan Fast Universal Master Mix and TaqMan probes (Applied Biosystems) or the Universal Probe Library (UPL, Roche) system. Gapdh or Actb were used as an endogenous control (Applied Biosystems). For siRNA and rescue experiments, the data were further normalised to control cell line. All experiments were performed in biological duplicate if not otherwise indicated. Results are shown as mean and standard deviation. TaqMan probes were Pou5f1 (Oct4), Mm00658129_gH; Klf4, Mm00516104_m1; Klf2, Mm01244979_g1; Nanog, Mm02384862_g1; Rex1, Mm03053975_g1; Pou3f1 (Oct6), Mm00843534_s1; and Fgf5, Mm03053745_s1. Primer sequences and UPL probe numbers are listed in Table EV2.