2.7. Gene expression analysis

Extraction of total RNA from each sample was conducted using RNAzol (Molecular Research Center, Cincinnati, OH, USA) based on manufacturer’s procedures. The concentration of each RNA sample was quantified with a spectrophotometer (NanoDrop^TM ND-1000, Thermo Fisher Scientific). Reverse transcription was performed using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV; Promega, Madison, WI, USA) to produce cDNA from total RNA. The RT-qPCR was done using a DyNAmo Hot Start SYBR Green qPCR kit (Thermo Fisher Scientific) to quantify the expression of MyoD, MyoG, mTOR, and a normalizer gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers for MyoD, MyoG, and GAPDH (Table 1) were previously designed, and the specificity confirmed as reported in Clark et al. [22]. Primers for mTOR were designed using primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and the specificity of the mTOR primers was confirmed by DNA sequencing the PCR product (Molecular and Cellular Imaging Center, The Ohio State University, Wooster, OH, USA). The RT-qPCR reaction was run in a DNA Engine Opticon 2 real-time machine (Bio-Rad) and included: denaturation for 30s at 94°C; annealing for 30 s at 58°C for MyoD and MyoG or at 55°C for GAPDH and mTOR; and elongation for 30 s at 72°C; and a final elongation at 72°C for 5 min in a DNA Engine Opticon 2 real-time machine (Bio-Rad). A standard curve of each gene was generated using serial dilutions of purified PCR products [70]. Arbitrary concentrations from 1 to 100,000 were assigned to each serial dilution. Since the concentration of each amplified cDNA sample was within the concentration range of the corresponding standard curve, the arbitrary molar concentration of each amplified sample was calculated according to the threshold cycle. The arbitrary molar concentration of each cDNA sample was normalized using GAPDH. The RT-qPCR for each gene was repeated in two independent cultures with 12 replicate wells per treatment group per cell line.

¹MYOD, Myogenic Determination Factor 1

²MYOG, Myogenin

³mTOR, Mechanistic Target of Rapamycin

⁴GAPDG, Glyceraldehyde-3-phosphate dehydrogenase

⁵bp, number of base pairs