Alternative Splicing of Key Genes in DM1 Myotubes

Total RNA from human myotubes was isolated using TRIreagent (Sigma). One microgram of RNA was digested with DNase I (Invitrogen) and reverse-transcribed with SuperScript II (Invitrogen) using random hexanucleotides; cDNA was used in a standard PCR reaction with GoTaq polymerase (Promega). Specific primers were used to analyze the alternative splicing of DMD, cTNT, SPTAN1, CAPZB, and DLG1 in control and DM1 human myotubes^{12 (link)}. GAPDH was used as endogenous controls. PCR products were quantified using ImageJ software (NIH). For multiplex qRT-PCR we used 1 ng of human myotube cDNA as template using the QuantiFast Probe PCR Kit reagent. Commercial TaqMan probes (Qiagen) were used to detect human MBNL1, MBNL2, and DMPK expression levels with an Applied Biosystems StepOnePlus Real-Time PCR System. Results from myotubes were normalized to GAPDH (VIC-labelled probe; Integrated DNA Technologies). Expression relative to the endogenous gene and control group was calculated using the 2^−∆∆Ct method. Exon inclusion data come from at least three biological replicates, while real-time PCR was done with three technical replicates from each of three independent biological samples.