Reverse transcription-quantitative real-time PCR (RT-qPCR)

Briefly, HepaRG cells were treated overnight in six-well dishes with either DMSO vehicle or 50 nM TBT, then total RNA was isolated using RNeasy mini kits (Qiagen). cDNA synthesis and qPCR using Roche Universal Library Probes was performed with comparative Ct data analysis as previously described, using β-actin mRNA as the normalizer [24]. The primers used for SREBF1 and FASN mRNA detection with Roche probes #77 and #11, respectively, are: SREBF1 Forward, 5’- cgctcctccatcaatgaca-3’; SREBF1 Reverse, 5’- tgcgcaagacagcagattta-3’; FASN Forward, 5’- caggcacacacgatggac-3’; FASN Reverse, 5’- cggagtgaatctgggttgat-3’. Student’s t test was used in Excel to define statistical significance of TBT gene inductions as compared to the DMSO control.