Quantification of Gene Expression in Liver and Muscle

Liver and skeletal muscle were collected and stored in RNAlater™ Stabilization Solution (Thermo Fisher Scientific, Waltham, MA, USA) and total RNA was extracted using the Trizol method, as described previously. The transcript levels of target genes and GAPDH were monitored by LightCycler^®480 Real-time PCR (Roche Diagnostics GmbH, Mannheim, Germany) using gene-specific primers (Eurofins, Bangaluru, India) as shown in Table A1. Results are reported as a relative expression normalized with the GAPDH housekeeping gene. The fold variation was determined using the 2^−ΔΔCt method according to previously published protocol [17 (link)].