Figure 3.

Confirmation of hSkM diurnal gene regulation by analysis of mSkM. Real-time RT-PCR (comparative C_T method) was performed on total RNA isolated from wild-type C57BL/6J mouse quadriceps muscle, collected at the indicated zeitgeber times (ZT). (a) Genes with a cycling phase similar to that of Bmal1, a key diurnal clock gene, are shown. (b) Genes with a cycling phase similar to that of Per1 and Per2. One-way ANOVA was applied to all time points and confirmed a statistically significant effect of time on gene expression levels (p < 0.001 for Bmal1 and Hat; p < 0.05 for all others). By normalizing average peak value to the average trough value (12-hour opposite peak), the following fold increases in gene expression were calculated: Bmal1 = 4.1, G0s2 = 7.0, Cry1 = 4.5, Nfil3 = 11.1, Per1 = 3.5, Per2 = 13.0, C/EBPb = 3.5, MyF6 = 3.3, Ier3 = 1.8, Hat = 1.8, and Gadd = 2.1. Values are mean 6 SEM. Gapdh was used as the reference gene and is included as the negative control.