Abi sequence detector software version 2

The real-time RT-PCR analysis was performed using an ABI7900HT Instrument (Applied Biosystems, Waltham, MA, USA). For the TaqMan analysis, predesigned or optimized assays on demand (Applied Biosystems) were used, including MITF (ID: Hs01117294_m1), tyrosinase (ID: Hs00165976_m1), TRP-1 (ID: Hs00167051_m1), TRP-2 (ID: Hs01098278_m1), 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. The real-time RT-PCR analysis was conducted as previously described [20 (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 the target genes were normalized to the levels observed in the controls. The results were verified by repeating each experiment four times in triplicate.

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.

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.