Quantitative RT-PCR Viral Gene Expression

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and samples (500 ng) mixed with RT-PreMix (Bioneer, Daejeon, Korea) and OligodT₁₈ (Bioneer, Daejeon, Korea) to synthesize cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using the Touch Real-Time PCR System (Bio-Rad, Hercules, CA) with qPCR Master Mix (Bioneer, Daejeon, Korea) and the following primers: HA (5′-ttgctaaaacccggagacac-3′ and 5′-cctgacgtatttgggcact-3′); NP (5′-gaatggtgctctctgcttttga-3′ and 5′-tccactttccgtttactctcctg-3′); PA (5′-aagtgccataggccaggtttc-3′ and 5′-cctcatctccattccccatttc-3′); M2 (5′-gaaaggagggccttctacgg-3′ and 5′-tcgtcagcatccacagcac-3′); canine IFN-β (5′-ccagttccagaaggaggaca-3′ and 5′-tgtcccaggtgaagttttcc-3′); canine Mx1 (5′-gaatcctgtacccaatcatgtg-3′ and 5′-taccttctcctcatattggct-3′); and canine β-actin (5′-tgccttgaagttggaaaacg-3′ and 5′-ctggggcctaatgttctcaca-3′) (Shoji et al., 2015 (link)). β-Actin expression was used as an internal reference, and relative expression of each gene was calculated using the ΔΔCt method. Every reaction was performed in triplicate.