Reverse transcription master mix

Total RNA was isolated from the same number of cells or similar tissue weight between experimental conditions. RNA was isolated with Trizol (Invitrogen, Waltham, MA, USA) and cDNA was synthesized from 2 μg of RNA using 2X Reverse Transcription master mix (Applied Biosystems, Waltham, MA, USA) following the manufacture’s protocol. The cDNA was quantified by real-time PCR with KAPA HiFi HotStart PCR Kit (Kapa Biosystems, Wilmington, MA, USA) using the LightCycler 480 System (Roche, Basel, Switzerland). Primer sequences used were as follows: ACTIN: fwd 5′-TACCACCATGTACCCAGGCA-3′, rev 5′-CTCAGGAGGAGCAATGATCTTGAT-3′; GAPDH: fwd 5′-CGCTCTCTGCTCCTCCTG-3′, rev 5′-CGATGGTGTCTGAGCGAT-3′; Mt1: fwd 5′-ATGGACCCCAACTGCTCCT-3′, rev 5′-ACAGCCCTGGGCACATTT-3′; Mt2: fwd 5′-CCGATCTCTCGTCGATCTTCAACC-3′, rev 5′-CAGGAGCAGCAGCTTTTCTTGCAG-3′; NFE2L1: fwd 5′-TGGAACAGCAGTGGCAAGATCTCA-3′, rev 5′-GGCACTGTACAGGATTTCACTTGC-3′; NFE2L2: fwd 5′-TTCCCGGTCACATCGAGAG-3′, rev 5′-TCCTGTTGCATACCGTCTAAATC-3′; MT2A: fwd 5′-CTCTTCAGCTCGCCATGGAT-3′, rev 5′-TGGAAGTCGCGTTCTTTACA-3′; MT1G: fwd 5′-TTGCAATGGACCCCAACT-3′, rev 5′-TCCTGGATTTTACGGGTCAC-3′; NQO1: fwd 5′-CAGCTCACCGAGAGCCTAGT-3′, rev 5′-AGTGCTCTTCTGCCGACCAT-3′.

Total RNA from cells was extracted with PRImeZOL (Canvax, #AN1100) and cDNA was synthetized using 2X Reverse transcription master mix (Applied Biosystems, Waltham, MA, USA) according to manufacturers’ protocols. For quantitative real-time PCR (RT-PCR), SYBR Green fluorescent reagent and LightCycler 480 Real-Time PCR System (Roche) were used. All RT-PCR were performed in experimental triplicate. Primer sequences are listed in Table EV1.

The reverse-transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The reverse transcription reactions were carried out in 20 µL volume; the input amount of total RNA was 1 µg diluted to a volume of 10 µL in nuclease-free water. A 2X Reverse-Transcription Master Mix was prepared according to Table 1 (Applied Biosystems, Foster City, CA, USA).
The reactions were carried out in a thermal cycler (Eppendorf Mastercycler) using the cycling conditions described in Table 2.