Antioxidant and Immune Gene Expression in Laying Hens

The liver tissues were randomly selected from three laying hens per replicate. The liver tissues were immediately removed and frozen at −20 °C until RNA extraction. Fifty milligrams of samples were mixed with lysis buffer and homogenized using a tissue homogenizer. RNA extraction was then carried out according to manufacturer’s protocol using a column-based RNA extraction kit (Invitrogen, PureLinkTM RNA Mini Kit, MA USA). The concentration and purity of total RNA was measured at an absorbance ratio of 260–280 nm using NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthesized using Bio-Rad iScriptTM RT Supermix cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The primers required for IL-1β, IL-6, IL-10, TNF-α, SOD, CAT, GSH-Px1, and Nrf2 genes were designed according to Table 2. Employing the CFX Connect^TM Real-Time PCR System (Bio-Rad, USA) and the iTaq Universal SYBR Green supermix 2× (Bio-Rad, USA) in addition to the specific primers for individual genes, the qPCR reaction was conducted. The expression levels of the antioxidant and immune-related gene were measured using the 2^−ΔΔCt method and a standard curve [23 (link)].