RNA isolation and quantitative real-time polymerase chain reaction

Cultured cells were superficially washed with PBS, followed by extraction of total RNA using Trizol (Invitrogen) according to the manufacturer's standard instructions. Samples (2 µg) of total RNA were reverse transcribed, followed by oligo (dT) primer and MMLV Reverse Transcriptase (Promega Co., Madison, WI, USA) at a final volume of 25 µL. Aliquots of 2 µL cDNA were used as templates for real-time polymerase chain reaction (PCR). PCR amplification was performed with 2×SYBR Premix Ex Ta (Takara Bio Inc., Shiga, Japan) and 10 pmol forward and reverse primers using Thermal Cycler DICE Real Time System (Takara Bio Inc.). Reactions were performed for 45 cycles of 95℃ for 10 seconds, 60℃ for 15 seconds, and 72℃ for 30 seconds. Primers are listed in Table 1.

NFE2L1, nuclear factor-E2-related factor 1; MT1, metallothionein 1; MT2, metallothionein 2; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; NQO1, NAD(P)H dehydrogenase, quinone 1; GPx1, glutathione peroxidase 1.