Real-Time PCR for mRNA Expression Analysis

To measure the level of mRNA expressions, real-time PCR was performed using the StepOnePlus system (Applied Biosystems, Foster City, CA) as described previously (Haraguchi et al., 2010 (link); Haraguchi et al., 2012b (link); Haraguchi et al., 2012b (link); Haraguchi et al., 2015 (link); Nozaki et al., 2018 (link)). The sequence of each primer is shown in Table 1. Gapdh was used as the internal standard. The reaction mixture contained SYBR Green Real-Time PCR Mix (Toyobo, Osaka, Japan), 400 nM of forward and reverse primers, and 30 ng of cDNA in a final volume of 20 μL. PCR was run with a standard cycling program: 95°C for 3 min; 40 cycles of 95°C, 15 s; 60°C, 15 s; and 72°C, 15 s. An external standard curve was generated by a serial 10-fold dilution of cDNA obtained from the salmon brain, which had been purified, and its concentration was measured. To confirm the specificity of the amplification, the PCR products were subjected to melting curve analysis and gel electrophoresis. The results were normalized to the expression of gapdh using StepOnePlus 2.0 software (Applied Biosystems).