RT-PCR Analysis of Nrf2, CYP1A1, and HO-1

Real-time polymerase chain reaction (RT-PCR) analysis was conducted as previously described [25 (link)]. Specifically, RT-PCR analysis was done with an ABI7900HT Instrument (Applied Biosystems, Waltham, MA, USA). Predesigned or optimized assays on demand (Applied Biosystems) were used for TaqMan analysis. They include Nrf2 (ID: Hs00975961_g1), CYP1A1 (ID: Hs01054796_g1), HO-1 (ID: Hs01110250_m1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ID: Hs00266705_g1), 18S (Hs03003631_g1), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Hs02800695_m1). ABI Sequence Detector Software version 2.0 (Applied Biosystems, Waltham, MA, USA) was used to analyze the data. TRI reagent^® was introduced to extract total RNA from the cells according to the manufacturer’s protocols and the total RNA was stored at –70 °C until use. MuLV reverse transcriptase was used to synthesize cDNA from the total RNA (1 μg) according to the manufacturer’s instructions. RT-PCR results was analyzed as previously reported [19 (link)]. The data were normalized to the expression level of three housekeeping genes such as GAPDH, 18S, and HPRT. The expression levels of target genes were normalized to the levels measured in the controls. The same experiment was repeated four times to verify the results and each experiment was conducted in triplicate.