Step one plus real time pcr machine

Cells were harvested in cold 1X PBS and RNA extraction was completed utilizing the TRIzol reagent (Thermo Fisher) according to standard protocol. Reverse transcription reactions of the RNA were performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem), as per the manufacturer’s protocol. The cDNA samples were then used as a template for qRT-PCR. Applied Biosystem’s Step One Plus Real-time PCR machine was used with BioRad’s iTaq^™ Fast SYBR^® Green Supermix with ROX according to the manufacturer’s protocol. The primer sets used for each gene can be found in S1 Table. GAPDH was used as the endogenous reference control. Statistical significance was measured by Student’s t test.

Total RNA was extracted from cells or liver tissues from human liver-chimeric mice using Trizol reagent (Solarbio, Beijing, China). First-strand cDNA was synthesized using the Hifair Ⅲ 1st strand cDNA synthesis supermix kit (Yeasen Biotech, Shanghai, China), in which the 5×gDNA digester Mix could remove the residual genomic DNA contamination. Quantitative real-time PCR was performed on StepOnePlus real-time PCR machine (Bio-Rad), using qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai, China). RT reaction without the enzyme followed by qPCR was used as a RT control in the experiments. Relative transcriptional folds were calculated as 2^-ΔΔCt. The primers used were listed in Table S6.