Quantitative RT-PCR
Total RNA was prepared following standard protocols. Briefly, each frozen sample was placed into a tube containing a 5-mm stainless steel bead. Working on ice, 1 mL Trizol reagent (Invitrogen, Groningen, The Netherlands) was added, and homogenisation was performed in a TissueLyser (Qiagen, Valencia, CA) at 30 Hz for 3 min twice. RNA was extracted with chloroform/isopropyl alcohol/ethanol and stored at –80°C until use. One μg of total RNA was used to synthesize cDNA using Iscript reverse transcriptase (BioRad Laboratories, Marnes La Coquette, France) and oligo-dT primer as specified by the manufacturer. Gene expression was measured using the SYBR green reagent (2× SYBR Green Supermix; Bio-Rad Laboratories) following the manufacturer's instructions on a Bio-Rad iCycler. PCR was performed in optimized conditions: 95°C denatured for 3 min, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. Primers (Eurogentec, Seraing, Belgium) were as follows: acetylcholine receptor α-subunit (AChR-α), forward 5′-ccacagactcaggggagaag-3′, reverse 5′-aacggtggtgtgtgttgatg-3′; acetylcholine receptor γ-subunit (AChR-γ), forward 5′-gagagccacctcgaagacac-3′, reverse 5′-gaccaacctcatctccctga-3′; acetylcholine receptor ε-subunit (AChR-ε), forward 5′-caatgccaatccagacactg-3′, reverse 5′-ccctgcttctcctgacactc-3′; muscle-specific receptor tyrosine kinase (MuSK), forward 5′-ttcagcgggactgagaaact-3′, reverse 5′-tgtcttccacgctcagaatg-3′; peroxisome proliferator-activated receptor α (PPARα), forward 5′-cacgcatgtgaaggctgtaa-3′, reverse 5′-gctccgatcacacttgtcg-3′; peroxisome proliferator-activated receptor γ, coactivator 1-α (PGC1-α), forward 5′-tgctgctgttcctgttttc-3′, reverse 5′-ccctgccattgttaagacc-3′; pyruvate dehydrogenase kinase, isoenzyme 4 (PDK4), forward 5′-cgcttagtgaacactccttcg-3′, reverse 5′-cttctgggctcttctcatgg-3′; ribosomal RNA 18S (18S), forward 5′-cgtctgccctatcaactttcg-3′, reverse 5′-ttccttggatgtggtagccg-3′; SCD1, forward 5′-cctacgacaagaacattcaatcc-3′, reverse 5′-cgtctcaagttctcttaatcct-3′. Relative quantification was achieved by calculating the ratio between the cycle number (Ct) at which the signal crossed a threshold set within the logarithmic phase of the gene of interest and that of the 18S reference gene. Ct values were the means of duplicates.