M mlv reverse transcriptase enzyme system

A GeneJET RNA Purification Kit (Thermo Fisher Scientific) was used to extract the total RNA from homogenized liver samples, according to the manufacturer’s protocol. 1 μg total RNA was then used to synthesize cDNA using a M-MLV Reverse Transcriptase Enzyme System (Life Technologies, Thermo Fisher Scientific) and OligoT primers. qPCR was performed using SYBR Green Master Mixes on a Quanta studio 3 PCR machine (Life Technologies–Applied Biosystems, Thermo Fisher Scientific), using gene-specific primers included in the Supplementary primer list Table 2. Gene expression was calculated using the 2^–ΔΔCt method and normalized to the housekeeping gene Actin.

Total RNA was isolated from liver tissues using a GeneTET RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. cDNA was synthesized using an M-MLV Reverse Transcriptase Enzyme System (Life Technologies, Thermo Fisher Scientific) and OligodT primers. The resulting cDNA products were subjected to qPCR reaction using SYBR Green Master Mixes. qPCR was performed on a Quanta studio 3 PCR machine (Life Technologies–Applied Biosystems, Thermo Fisher Scientific). The threshold crossing value(Ct) was determined for each transcript and then normalized to that of the internal gene transcript(β-actin). Fold change values were then calculated using the 2^–ΔΔCt method. Genes-specific primers were designed using Integrated DNA Technologies (IDT) PrimerQuest software. Sequences of the forward and reverse primers are listed in Supplementary Table S7.