Total RNA was extracted from 20 mg of liver specimens via a centrifugal column method (Beijing Tsingke Biotech Co., Ltd.). Using a Nanodrop 2000 ultra microvolume spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), RNA purity was analyzed. An optical density 260/280 ratio of approximately 2.0 indicated high purity.
A SynScript®Ⅲ RT SuperMix for qPCR kit was subsequently used to prepare first-strand cDNA. The mixture of RNA and kit reagents was subjected to 15 min of incubation at 50 °C and 5 s at 85 °C to synthesize cDNA.
A quantitative polymerase chain reaction (qPCR) assay was performed using an ArtiCanATM SYBR qPCR kit and an ABI Quantstudio6 Flex Real-Time PCR System (Applied Biosystems Inc., Singapore). qPCR was conducted using specific primers (Table 1 ) and involved the following steps: 1 min initial denaturation at 95 °C; 10 s denaturation at 95 °C, 20 s annealing at 60 °C, and 1 min final extension at 72 °C for 40 cycles. GAPDH served as an endogenous control. Utilizing the cycle threshold (Ct) value, relative gene expression was determined through the 2−∆∆CT methodology.
A SynScript®Ⅲ RT SuperMix for qPCR kit was subsequently used to prepare first-strand cDNA. The mixture of RNA and kit reagents was subjected to 15 min of incubation at 50 °C and 5 s at 85 °C to synthesize cDNA.
A quantitative polymerase chain reaction (qPCR) assay was performed using an ArtiCanATM SYBR qPCR kit and an ABI Quantstudio6 Flex Real-Time PCR System (Applied Biosystems Inc., Singapore). qPCR was conducted using specific primers (