4.7. qRT-PCR Gene Expression Assay

To analyze the expression of cytokines, we designed primers using Lasergene software (DNASTAR, Madison, WI, USA) (Table 1) and performed qRT-PCR using 2× Power SYBR^® Green Master Mix (Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions, with the LightCycler^® 96 system (Roche). The cycle number when the fluorescence first reached a preset threshold was used to quantify the initial concentration of the individual templates for the expression of the mRNA of genes of interest. Chicken GAPDH was used as an internal control gene to normalize for RNA quantity. The relative gene-specific expression was calculated using the 2^−ΔΔCt method after normalization to GAPDH [52]. All qRT-PCRs were performed in triplicate.