Total RNA was isolated from the treated cells using a
mirVana™ miRNA Isolation Kit (Life Technologies, Palo Alto, CA, USA). Total RNA was then reverse-transcribed to cDNA using a
ReverTra qPCR RT Kit (Toyobo, Osaka, Japan). Each reaction was run using
Thunderbird SYBR qPCR Mix (Toyobo) on a
Light Cycler 1.5 (Roche, Indianapolis, IN, USA). The comparative Ct method was used to determine the fold changes in the expression using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. Each sample was run in triplicate. Primers were used for Nrf2 (forward, 5′-AGTGGATCTGCCAACTACTC-3′; reverse, 5′-CATCTACAAACGGGAATGTCTG-3′), malic enzyme 1 (ME1; forward, 5′-CTGCCTGTCATTCTGGATGT-3′; reverse, 5′-ACCTCTTACTCTTCTCTGCC-3′), glutamate-cysteine ligase catalytic subunit (GCLC; forward, 5′-TGAAGGGACACCAGGACAGCC-3′; reverse, 5′-GCAGTGTGAACCCAGGACAGC-3′); glutamate-cysteine ligase modifier subunit (GCLM; forward, 5′-AATCTTGCCTCCTGCTGTGTGA-3′; reverse, 5′-TGCGCTTGAATGTCAGGAATGC-3′), and GAPDH (forward, 5′-CAACAGCCTCAAGATCATCAGC-3′; reverse, 5′-TTCTAGACGGCAGGTCAGGTC-3′). The cycling conditions consisted of initial denaturation at 98 °C for 5 min followed by 45 cycles at 98 °C for 15 s, 58 °C for 30 s, and 72 °C for 60 s. These experiments were performed in triplicate.
Matsuoka Y., Yoshida R., Kawahara K., Sakata J., Arita H., Nkashima H., Takahashi N., Hirayama M., Nagata M., Hirosue A., Kuwahara Y., Fukumoto M., Toya R., Murakami R, & Nakayama H. (2022). The antioxidative stress regulator Nrf2 potentiates radioresistance of oral squamous cell carcinoma accompanied with metabolic modulation. Laboratory Investigation; a Journal of Technical Methods and Pathology, 102(8), 896-907.
