Quantitative Real-Time PCR Protocol

Total RNA was extracted using Trizol reagent (for feather) or Trizol LS (for blood) (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. RNA integrity and quality was assessed using 1% agarose gel electrophoresis and RNA concentrations and purity were determined for each sample by Take 3 Micro-Volume Plate using Synergy HT multi-mode micro plate reader (BioTek, Winooski, VT). RNA samples were DNase treated, reverse transcribed using qScript cDNA Synthesis Supermix (Quanta Biosciences, Gaithersburg, MD), and amplified by real-time quantitative PCR (Applied Biosystems 7500 Real Time System) with PowerUp SYBR green master mix (Life Technologies, Carlsbad, CA, United States) as previously described (Rajaei-Sharifabadi et al., 2017 (link)). The qPCR cycling conditions were 50°C for 2 min, 95°C for 10 min followed by 40 cycles of a two-step amplification program (95°C for 15 s and 58°C for 1 min). At the end of the amplification, melt curve analysis was applied using the dissociation protocol to exclude contamination with unspecific PCR products. Relative expression of the target genes was determined using the 2^–ΔΔCt method, with normalization to 18 s rRNA as a housekeeping gene (Schmittgen and Livak, 2008 (link)). Oligonucleotide primer sequences specific for chicken are presented in Table 2.