Realtime PCR
Real time PCR was carried out according to methods previously described (Ahuja, Kim, Sung, & Cho, 2020; Wu et al., 2022). DNA free RNA was extracted from the C2C12 myotubes using zymo quick RNA mini kit (R1055) from with DNase treatment and RNA from tissues were isolated using Trizol (Invitrogen) according to manufacture protocol. Briefly 1ug of total RNA was reverse transcribed using first strand cDNA synthesis kit (thermos fisher) real time PCR was performed in triplicate using Bio-Rad icycler following manufacture protocols. The relative gene expression of the target genes was calculated by using 2-∆∆ct method (Qiu et al., 2021), the values were normalized to the housekeeping gene GPADH. Primer sequence of the respected primers are mentioned in table no1.