Cfx 96 connect optics module

Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed using a PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China). Real-time PCR was performed in a CFX 96 Connect™ Optics Module (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green PCR Master Mix (Takara Biotechnology). Aliquots of cDNA were used for PCR using primer sets specific to Nrf2, GCLC, HO-1 and NQO1, and GAPDH as a control. Primers were as follows: Nrf2: 5′-AGCGACGGAAAGAGTATGA-3′, 5′-TGGGAGTAGTTGGCAGAT-3′; GCLC: 5′-TTGTTATGGCTTTGAGTG-3′, 5′-TCTGAGTTTGGAGGAGGG-3′; HO-1: 5′-AATGGTTCAGGCAACAGGG-3′, 5′-CTCCAGCAGTATGAGCAAAGTA-3′; NQO1: 5′-TGGTTTGAGCGAGTGTTC-3′, 5′-TATTCTCCAGGCGTTTCT-3′; GAPDH: 5′-GGTGAAGGTCGGAGTCAACGGA-3′, 5′-GAGGGATCTCGCTCCTGGAAGA-3′. Data were analyzed according to the 2^−ΔΔct method, as previously described [53 (link)].

Total RNA was isolated from cells using TRIzol reagent [17 (link)]. cDNA was synthesized using a kit from Takara Bio Inc. following the manufacturer’s instructions, and reverse transcribed according to the instructions of the Prime Script RT reagent kit (Takara Biotechnology Co., Ltd., Beijing, China). Aliquots of cDNA were subjected to qPCR analysis with SYBR Green PCR Master Mix (Takara Biotechnology Co., Ltd., Beijing, China) and a real-time PCR system equipped with a CFX 96 Connect™ Optics Module (Bio-Rad Laboratories, Inc., Hurcules, CA, USA). The reaction program was predenaturation for 3 min at 95 °C, then 40 cycles of denaturation at 95 °C for 5 s, annealing at 54 °C for 15 s, and extension at 72 °C for 30 s. Tubulin was used as the internal reference gene. The PCR primer sequences used are shown in Table 1. Data were analyzed using the 2^−∆∆cq method [18 (link)].