RNA extraction
Liver tissue RNA was extracted by Trizol method. In brief, Approximately 50 mg of liver tissue was firstly placed in a mortar(Thermofisher, USA), ground into a powder with liquid nitrogen and transferred to an enzyme-free tube and 1ml TRIZOL(Invitrogen, USA) reagent was added to the homogenization to lyse sample for 5 minutes at room temperature in the dark. Then, the 200μl of chloroform per was added and shake tubes vigorously by hand for 15s. The sample was allowed to stand at room temperature for 3 minutes, and centrifuged at 12, 000 g for 10 min at 4°C. Pipette the supernatant into another new enzyme-free tube, 400 μl Isopropanol was added and shake tubes slightly by hands for 15s. The sample was allowed to stand at room temperature for 3 minutes, and centrifuged at 12, 000 g for 10 min at 4°C. Discard the supernatant, added 1ml 75% ethanol into tube and shake tube for 30s. centrifuged at 7, 500 g for 5 min at 4°C. Then, discard the supernatant and let the alcohol evaporate naturally. Finally, RNA pellet was redissolved with the 20μl Enzyme-free water. The RNA degradation and contamination were detected with 1% agarose gel test and RNA concentration was detected by NanoDrop Microvolume Spectrophotometers(Thermofisher, USA).