*4.6. Quantative Real-Time PCR*

Portions of the right gastrocnemius muscle of mice, inguinal WAT, and interscapular BAT were processed by using a tissue homogenizer (Omni International, Kennesaw, GA, USA). Total RNA from tissue homogenate was isolated using TriZol (Life Technology, Carlsbad, CA, USA). Total RNA (3 μg) was reverse transcribed to cDNA with SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA, USA). Quantitative real-time RT-PCR of target genes was performed using KAPA SYBR FAST qPCR kit (KAPA Biosystems, Wilmington, MA, USA) [23]. Glyceraldehyde−3-phosphate dehydrogenase (GAPDH) was used as an internal control. Expression levels were calculated according to the relative 2−ΔΔCt method. All primers are listed (Supplemental Table S2).
