Gotaq quantitative pcr qpcr master mix

Total RNA extraction was performed with TRIzol (Life Technologies, USA) according to the manufacturer's instructions, and then RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used for reverse transcription. Following, GoTaq® quantitative PCR (qPCR) Master Mix (Promega, USA) was used for quantitative PCR with indicated primers on a VIIA(TM) 7 System (Applied Biosystems). Data were analyzed by normalization against GAPDH. The primers used are indicated as in Table 1.

Total RNA was isolated from HEK293T cells at 3 d of cultivation by using Trizol reagent (Thermo Fisher Scientific). RNA was reverse transcribed by using the SuperScript II First-Strand Synthesis System (Thermo Fisher Scientific). Quantification of NRF2 and AMPK mRNA was performed by using the GoTaq Quantitative PCR (qPCR) Master Mix (A6001; Promega, Madison, WI, USA). Forward and reverse primer sequences used for qPCR were as follows: NRF2: 5′-TCCAGTCAGAAACCAGTGGAT-3′and 5′-GAATGTCTGCGCCAAAAGCTG-3′; AMPK: 5′-ACTGTACCAGGTCATCAGTACACC-3′and 5′-TCCAGGTACATCAGATTTCCTTC-3′; and GAPDH: 5′-TGCACCACCAACTGCTTAGC-3′ and 5′-GGCATGGACTGTGGTCATGAG-3′. The PCR thermal program included the following: 10 min at 95°C (1 cycle), and 30 s at 95°C, 45 s at 58°C, 30 s at 72°C (40 cycles), 5 min at 95°C, 1 min at 55°C, 30 s at 97°C (1 cycle). Measures were performed on an Mx3005P qPCR System (Stratagene, San Diego, CA, USA). Melting curve analysis was performed to confirm correct PCR product size and absence of nonspecific bands. Relative mRNA levels were determined by normalizing the PCR threshold cycle number of NRF2 and AMPK with that of the GAPDH reference gene. Normalized data were analyzed by using independent 2-sample Student’s t tests with Bonferroni correction (SPSS v.20 software; IBM, Armonk, NY, USA).