Time course of C2C12 differentiation. (A) C2C12 cells were placed in differentiation medium when culture reached 80% of confluence (t = 0 h). After 48 hours, the first myotubes, indicated by arrows, had clearly formed and after 11 days (t = 264 h), most of the cells had merged into myotubes. C2C12 RNA was extracted at several differentiation time points and tested for the presence of (B) mRNA from the myogenic markers Myf5, MyoD, Myogenin and Mrf4 and (C) mRNA from the muscle-specific markers Csrp3, Hes6, Mef2a and Mef2d. Transcription levels are expressed as relative quantities (RQ) compared to the initiation of differentiation (t = 0 h). The grey area includes no significant variations (RQ < ± 2). For (B), standard deviations were calculated on three separate experimental values. No significant transcriptional expression of Mrf4 was detected before t = 192 h (Ct>33, dotted line). The top inset presents the standard expression patterns of the four myogenic markers Myf5, MyoD, Myogenin and Mrf4 as found in the literature.