Low rox and taqman qpcr probes

2D cultured HepG2 and primary human hepatocytes and 3D seeded scaffolds were digested with Trizol TRI Reagent (Sigma-Aldrich, St Louis, MO, USA) and RNA was extracted following the manufacturer’s instructions. Precipitated and dried RNA was re-suspended in nuclease free water (Qiagen, Hilden, Germany). RNA concentration was measured using Nanodrop1000 (ThermoScientific, Waltham, MA, USA). RNA was converted into cDNA using GoScript™ Reverse Transcriptase kit (Promega, Southampton, UK) according to the manufacturer’s protocol. cDNA concentration was adjusted to 10 ng/µL. Quantitative (q)PCR was performed on 25 ng of cDNA using PCR master mix (PrecisionPLUS-R—Primerdesign Ltd. Chandler’s Ford, UK) with low-ROX and Taqman qPCR probes (Integrated DNA Technology, Coralville, Ia, USA. List of probes in Supplementary Table S2) in MicroAmp Fast Optical 96 well Reaction Plates (Starlab, Milton Keynes, UK) using the ABI7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Target genes are reported in Table S3.

Cultured cells were collected for gene expression analysis in Lysis buffer+Thyoglycerol buffer from ReliaPrep™ kit (Promega) following the manufacturer’s instructions. Precipitated and dried RNA was re-suspended in nuclease free water (Qiagen). RNA concentration was measured using Nanodrop1000 (ThermoScientific). RNA was converted into cDNA with GoScript™ Reverse Transcriptase kit (Promega) according to the manufacturer’s protocol. cDNA concentration was adjusted to 10ng/μl. Quantitative (q)PCR was performed using PCR master mix (PrecisionPLUS-R, Primerdesign Ltd) with low-ROX and Taqman qPCR probes (Integrated DNA Technology, Table S3) in MicroAmp Fast Optical 96 well Reaction Plates (Applied Biosystems) using the QuantStudio 3 Real-Time PCR System (Applied Biosystems).