Gene expression changes induced by methylchrysenes in HepG2 cells

Quantitative polymerase chain reaction (qPCR) of target genes was used to test for induced gene expression in HepG2 cells treated with chrysenes and other compounds [22]. HepG2 cells were exposed to different chemicals for 6 h over a range of concentrations ranging from non-cytotoxic to modest toxicity. RNA was extracted from the cells using Qiagen’s RNeasy Plus Mini Kit (#74136) and following the manufacturer’s instructions. RNA purity and concentration were assessed using a Thermo Scientific Nanodrop 2000c spectrophotometer followed by dilution to 0.5 µg/µl in nuclease-free water to avoid RNA damage. Complementary DNA (cDNA) was synthesized using BioRad iScript cDNA synthesis kit (#170-8891) following the manufacturer’s instructions. cDNAs were stored at -20 degree C until use. Gene expression was detected using primer-probe sets to AhR-regulated genes from Applied Biosystems’ TaqMan Gene Expression Assays along with hypoxanthine phosphoribosyl transferase 1 (HPRT1) as a reference transcript. RT-PCR was conducted using a BioRad C1000 thermal cycler supplied with a CFX96 Real-Time PCR Detection System.