Kod sybr green chemistry

On days 3 and 7, cells were washed with 0.5 ml/well of PBS(−), and RNA samples were collected with 0.35 ml/well of Trizol (Thermo Fisher Scientific, United States). RNA extraction was performed using a silica column-based RNA extraction kit (Zymo Research, United States), and the quality and quantity of total RNA were measured by NanoDrop (Thermo Fisher Scientific, United States). Total RNA was converted to cDNA with a PrimeScript reverse transcriptase kit (Takara, Japan). The expression levels were assessed by quantitative real-time PCR using KOD SYBR Green chemistry (Takara, Japan) in the StepOnePlus (Applied Biosystems, United States) system. The list of genes measured is shown in Supplementary Table S2 with sequences of primers which were designed with Primer-BLAST and purchased from Eurofins Genomics. The expression levels were normalized by that of β-actin as an internal control.

Relative gene expression of hepatocyte phenotypes was assessed by quantitative real-time PCR. First, hepatocyte sandwich cultures were lysed in an appropriate amount of Trizol reagent (Thermo Fisher Scientific, USA), followed by RNA extraction using a silica column (Zymo Research, USA). Obtained total RNA was then quantified by Biospec-nano microvolume measurement (Shimadzu Scientific, Japan) and converted to complementary DNA with a PrimeScript reverse transcriptase kit (Takara, Japan). Individual primer sequences (Table 3) were designed with Primer-BLAST and purchased from Eurofins Genomics. Real-time PCR was performed with KOD SYBR Green chemistry (Takara, Japan) in a StepOnePlus (Applied Biosystems, USA) system, using β-Actin as an internal control. For detailed screening of cellular drug metabolism, RT² qPCR Rat Drug Metabolism Panels, targeting 84 different related genes, were used with RT² SYBR Green chemistry (Qiagen, Germany) according to manufacturer's recommendations. Similar to the analysis of individual targets, β-Actin was used as the reference gene.