Extraction and Quantification of High-Quality RNA

Liver samples were homogenized using an Ultraturax homogenizer (D1000 handheld homogenizer; Benchmark Scientific, Inc., Sayreville, NJ, USA). The muscle tissues were homogenized by grinding them in liquid nitrogen in a cooled mortar and pestle. Total RNA was isolated using TRIzol reagent according to the Direct-zol RNA Miniprep (R2052; Zymo Research Orange, CA, USA) kit protocol. The quantity and purity of RNA were determined using a microplate reader (Synergy HT Multi-Mode Microplate Reader-SN 1712214, BioTek Instruments, Inc., Winooski, USA) as previously described^{24 (link)}. The quality and integrity of RNA were checked using a Qubit RNA IQ assay kit (# Q33222, Thermo Fisher Scientific) measured using a Qubit 4 fluorometer (Invitrogen, Thermo Fisher Scientific). The RNA IQ numbers ranged from 8.7 to 10. cDNA synthesis was performed using the LunaScript RT SuperMix Kit (New England Biolabs Inc. E3010L) with 200 ng total RNA and a PCR^max Alpha Thermal Cycler (Cole-Parmer Ltd. UK). The cDNA was stored at – 80 °C until the RT-PCR assay.