*2.4. Real-Time qPCR Analysis*

Primers (Table 2) were designed by Primer Premier 5 [14] software based on the gene sequence of the mackerel tuna genome (Non-public data). GAPDH was used as the housekeeping gene. The qPCR was conducted with the Real-time qPCR analysis (Analytik Jena GmbH, Jena, Germany) using SYBR Green (Tiangen Biotech Co., Ltd., Beijing, China). The 20 μL of reaction, including 10 μL of 2× RealUniversal PreMix, 0.6 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template and 6.8 μL of RNase-free ddH2O. Reaction conditions were: (1) Pre-denaturation at 95 ◦C for 15 min; (2) Amplification reaction, denaturation at 95 ◦C for 10 s, annealing at 56 ◦C for 20 s, extension at 72 ◦C for 30 s and 40 cycles. There were three repetitions of each test. The dissociation curves were analyzed to ensure only specific products were obtained with no formation of primer dimers in each reaction. At the end of the reaction, the relative expression level of the target gene was calculated using the 2−ΔΔCT method [16]. The reaction efficiency was 90–110%, and Pearson's coefficients of determination (R2) were >0.97.

**Table 2.** Relevant primer information.



#### **Table 2.** *Cont.*
