Myogenic gene analysis

After 14 days of culture in induction medium or control medium, total RNA from different cultures were isolated using the RNeasy Mini kit (Qiagen, Valencia, CA). Reverse transcription (RT) was performed using 8 pg of total RNA with RT reaction mixture, which contains primers specific for target genes that were purchased from sigma and normalized to GAPDH as an endogenous control. The myogenic genes including α-SMA, calponin, SM-MHC were examined. Reverse transcription-polymerase chain reaction (RT-PCR) reactions were performed on StepOnePlus RT-PCR system (Applied Biosystems, Foster City, CA), using SYBR1 Green PCR Master Mix. The amplification reactions were carried out for up to 40 cycles. Fold variation in gene expression was quantified using the comparative Ct method: 2^{−Δ(ΔCtTreatment−ΔCtControl)}.