Total RNA was extracted from 10–20 individuals from each line using TRIzol (ambion, Beijing, China) following the manufacturer’s instructions. RNA was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit (Takara, Beijing, China). Gene expression analysis was performed by qRT-PCR using TB Green® Premix Ex Taq™ II FAST qPCR (Takara, Beijing, China), according to the manufacturer’s protocol in the CFX Connect Real-Time PCR Detection System (Bio-Rad, Beijing, China) through the Bio-Rad Manager ™ Software Version 3.1. Relative mRNA expression levels were normalized to that of Rpl32.
Primers used for qPCR are listed in Table 3. The primers for galbut virus and reovirus were designed by this study. The primers for galbut virus and La Jolla virus were from Webster C.L [12 (link)], and the primers for DCV were from Chuan Cao [14 (link)]. The primers for hap23sv were obtained from Chi-Wei Tsai [15 (link)].
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