2.5. Determination of Oxidative and Antioxidative Genes by qRT-PCR

Nrf2, catalase, glutathione peroxidase (gpx) 1 and 2, superoxide dismutase (sod) 1 and 2, and NADPH-oxidase subunits p47^phox and gp91^phox mRNA levels were detected using the Eco real-time PCR System (Illumina, San Diego, CA, USA) and Fast SYBR^® Green Master Mix (Applied Biosystems, Waltham, MA, USA). ARN purification was performed with Tri-Reagent (Merk, Kenilworth, NJ, USA) of PECs obtained from BALB/c mice at 20 dpi, SnPP-treated or untreated, as previously described [17]. Standard amplification conditions were 10 min at 95 °C, 40 thermal cycles of 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C, with a final extension of 10 min at 72 °C. The following primers were used: nrf2-F: 5′-CAGCATGTTACGTGATGAGG-3′, nrf2-R: 5′-GCTCAGAAAAGGCTCCATCC-3′, gpx1-F: 5′- GGGACTACACCGAGATGAACGA-3′, gpx1-R: 5′-ACCATTCACTTCGCACTTCTCA-3′, gpx2-F: 5′-GAGGAACAACTACCCGGGACTA-3′, gpx2-R: 5′-ACCCCCAGGTCGGACATACT-3′, sod1-F: 5′-TGGGTTCCACGTCCATCAGTA-3′, sod1-R: 5′-ACCGTCCTT TCCAGCAGTCA-3′, sod2-F: 5′-ATTAACGCGCAGATCATGCA-3′, sod2-R: 5′-TGTCCCCCACCATTGAACTT-3′, catalase-F: 5′-GCGTCCAGTGCGCTGTAGA-3′, catalase-R: 5′-TCAGGGTGGACGTCAGTGAA-3′, p47phox-F: 5′-GAGGCGGAGGATCCGG-3′, p47phox-R: 5′-TCTTCAACAGCAGCGTACGC-3′, gp91phox-F: 5′-CCAGTGAAGATGTGTTCAGCT-3′, gp91phox-R: 5′-GCACAGCCAGTAGAAGTAGA-3′, gapdh-F: 5′-ATGACATCAAGAAGGTGGTGAAG-3′, gapdh-R: 5′-TCCTTGGAGGCCATGTAGG-3′. Results were expressed as the ratio between each gene under study and GAPDH expression. Relative gene expression levels were calculated using the 2^−ΔΔCT method and normalized to GAPDH. All reactions were performed with at least five biological replicates.