2.7. Quantitative RT-PCR (qRT-PCR) for Antioxidant-Associated Genes

Total RNA, prepared by Trizol reagent (Invitrogen, Carlsbad, CA, USA), was reverse- transcribed to cDNA using the OmniScript RT kit (Qiagen, Valencia, CA, USA) as described previously [25]. qRT-PCR was performed by iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) using a MyiQ real-time machine (Bio-Rad). Touch-down PCR program [26] was used for the antioxidant-associated genes [27], including nuclear factor erythroid 2-like 2 (NFE2L2), glutathione-disulfide reductase (GSR), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione peroxidase 1 (GPX1), thioredoxin (TXN), catalase (CAT), superoxide dismutase 1 (SOD1), heme oxygenase 1 (HMOX1), NAD(P)H quinone dehydrogenase 1 (NQO1), and GAPDH. Their primer and PCR amplicon information are provided in Table 1. The comparative method (2–ΔΔCt) was used for analyzing relative mRNA expression (fold activation) [28].

Primer information for antioxidant-associated genes *.

* Primers without reference were designed in this study.