Quantstudio 7 flex real time rcr system

The total RNA was extracted using the miRNeasy Mini Kit (Qiagen, 217004) with RNase-Free DNase Set (Qiagen, 79254) according to the manufacturer’s instructions. A 10pg of mirVana miRNA mimic miR-199a-5p (ThermoFisher, Assay ID MC10893 and 002304) was added to 0.1–0.5ug of the total RNA as a spike-in control for RT-qPCR normalization. Reverse transcription was performed using PrimeScript™ RT Master Mix (TaKaRa, RR036A) for novel miRNA and TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems, 4366597) for the candidate miRNAs let-7b-5p (ThermoFisher, Assay ID 000378) and miR-34a-5p (ThermoFisher, Assay ID 000426). One microliter of cDNA was used for a real-time PCR using TaqMan™ Universal PCR Master Mix (Applied Biosystems, 4364340) on the ABI QuantStudio 7 Flex Real-Time RCR system. Primers and probes used are listed in the Additional file 5: Table S4. Results were calculated with 2(−ΔΔCt).

Reverse transcription was performed using total RNA and High Capacity RNA-to-cDNA (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Gene expression was measured using the QuantStudio 7 flex Real Time RCR System (Applied Biosystems, CA, USA), and the following primer and probe sets were used for the detection of each gene transcript: ALB (Hs00609411_m1), AFP (Hs00173490_m1), CYP1A2 (Hs00167927_m1), CYP2B6 (Hs04183483_g1), CYP2C9 (Hs00426397_m1), CYP2C19 (Hs00426380_m1), CYP2D6 (Hs00164385_m1), CYP2E1 (Hs00559368_m1), CYP3A4 (Hs00430021_m1), CYP7A1 (Hs00167982_m1), FXR (Hs01026590_m1), OATP1B1 (Hs00272374_m1), NTCP (HS00161820_m1), MRP2 (Hs00960489_m1), and BSEP (Hs00994811_m1). Gene expression levels are presented as relative values to the human liver-derived RNA levels (BioChain Institute, Inc., Newark, CA, USA). In addition, the expression level of CYPs in 22 lots of human cryopreserved hepatocytes shown in Table S1 was measured for comparison.