Gene Expression Analysis by qPCR

All qPCR analyses were run on a Stratagene Mx3000P Real-Time System (Agilent Technologies, United States) using SYBR Green I with ROX as reference dye. The reactions were performed with 10 μL of mixture consisting of 5 μL of 2XMaxima^TM SYBR Green qPCR Master Mix (Thermo Fisher, United States), 0.5 μL of cDNA and 100 nM primers (Integrated DNA Technologies, United States). Thermal conditions were as follows: 10 min at 95°C, 40 cycles of 15 s at 95°C, 20 s at 57°C, and 30 s at 72°C. Fluorescence was read after each annealing and extension phase. All runs were followed by a melting curve analysis from 55 to 95°C. Ubiquitin (UBI) was used as a reference gene to normalize the results. Primers for RBOH, CuZnSOD, MnSOD, FeSOD, CAT, GST, ICL, and UBI used in this work are reported in Supplementary Table S1. All amplification plots were analyzed with the MX3000P^TM software (Agilent Technologies, United States) to obtain Ct values. The relative expression levels of each gene were estimated using the method previously described by Pfaffl (2001).