Real-time RT-PCR analysis was performed using an ABI7900HT Instrument (Applied Biosystems, Waltham, MA, USA). For TaqMan analysis, predesigned or optimized assays on demand (Applied Biosystems) were used, including Nrf2 (ID: Hs00975961_g1), NQO1 (ID: Hs01045993_g1), AhR (ID: Hs00169233_m1), CYP1A1 (ID: Hs01054796_g1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ID: Hs00266705_g1), hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Hs02800695_m1), and 18S (Hs03003631_g1). The data were analyzed using ABI Sequence Detector Software version 2.0 (Applied Biosystems). Total RNA was extracted from cells using TRI reagent® according to the manufacturer's instructions and stored at -70°C until use. cDNA was synthesized from total RNA (1 μg) using MuLV reverse transcriptase according to the manufacturer's instructions. Real-time RT-PCR analysis was conducted as previously described [18 (link)]. The results were normalized to the expression level of endogenous GAPDH and were also tested against two additional housekeeping genes (18S and HPRT). We found that the results were not significantly different from those obtained using GAPDH. Expression levels of target genes were normalized to the levels observed in controls. Results were verified through four-time repetition of the same experiment, each of which was conducted in triplicate.
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