Quantitative PCR Analysis of BCAA Metabolizing Enzymes

mRNA levels of BCAA metabolizing enzyme related genes in SAT and AA tissues were measured by quantitative PCR by employing optimal reference gene pairs which was validated as previously described (39 (link)). Primer information used for the study are provided in Table 8. Powdered tissue samples were homogenized in Ribozol (N580-CA, Amresco, OH, USA). RNA was isolated as per the manufacturer's instructions and QIAxcel Advanced System (Qiagen, Toronto, ON) was used to determine the RNA quality and quantity. One microgram of RNA of was used to synthesize cDNA using qScript cDNA supermix (CA101414-104, Quanta Biosciences). qPCR analysis was performed using PerfeCTa SYBR green Supermix Low ROX (Quanta Biosciences, MA, USA) and a ViiA7 real-time PCR machine (Thermo Fisher Scientific, CA, USA) as detailed previously (39 (link)). qBase + software (Biogazelle) was used to quantify mRNA expression (39 (link)).