qPCR Analysis of Antioxidant Genes

The expression of Atf4, Chac1, Chop, Gclc, Gclm, Hmox1 and Nrf2 (Nfe2l2) genes was monitored by qPCR as previously described [23 (link)] with slight modifications. Briefly, the qPCR reactions were performed in duplicate in a final volume of 20 µL using TB Green PreMix ex Taq II Master Mix (Takara Bio Europe, France) and 200 nM primers (Table 1), in a RotorGene 6000 instrument (Corbett life science, Sydney, Australia). The amplification conditions were 95 °C for 10 min, 40 cycles at 95 °C for 10 s and 60 °C for 50 s. To confirm the absence of non-specific products or primer dimers, a melting curve analysis was performed from 65 to 95 °C at the end of each run, with a slope of 1 °C/s, and 5 s at each temperature. A duplicate non-template control was included for each target as a negative control. Gapdh (glyceraldehydes-3-phosphate dehydrogenase) and Gusb (β-D-glucuronidase) were used as reference genes. The relative expression levels were calculated using the 2^−ΔΔCt method [24 (link)].