Fig. 3
a and b. Expression of muscle gene transcripts in myocytes by subphenotype in early (48 h) and late (5 days) differentiation models. RNA was extracted from untreated control MG (n = 6) and OP-MG (n = 10) myocytes after 48 h and 5 days of differentiation as described. For each differentiation time point, expression levels of CHRNA1, MYOD1 and MYOG target genes were determined using relative quantification (2-∆Cq) where ∆Cq represents target gene Cq – average GUSB/TFRC Cq (the reference genes which were not influenced by prolonged differentiation of myocytes). a Combined log2 fold change for both subphenotypes (mean 2-∆∆Cq, where ∆∆Cq represents 5 days ∆Cq - 48 h ∆Cq) were compared to assess differences in gene expression levels between the early and late differentiation models. b Comparison of gene expression levels (2-∆Cq) between subphenotypes in the early and late differentiation models. c CHRNA1 P3A+ isoform expression in OP-MG and control MG myocytes represents in vivo muscle splicing signatures. RNA was extracted from control MG (n = 6) and OP-MG (n = 10) myocytes after 5 days of differentiation as described. qPCR was performed using two sets of primers for CHRNA1: 1 set which recognizes total CHRNA1 transcripts (P3A+ and P3A-) and another which is specific for P3A+ transcripts. Cq values were used to interpolate absolute transcript numbers from standard curves, then the ratio of P3A+:(P3A+ and P3A-) was calculated for each sample (expressed as %). Error bars show mean and SEM. Student’s t test was used for comparisons where the data was normally distributed, otherwise Mann-Whitney test was used (†) where Shapiro-Wilk normality test p < 0.05