Quantitative Analysis of wsv191 and wsv407 mRNA

To determine the mRNA level of wsv191 and wsv407, quantitative real-time PCR was performed with the wsv191-specific primers (5′-TTGACGAGGAGGATTGTAA AGG-3′ and 5′-ATACCAGGGTTTATTTTGTTGCG-3′) or wsv407-specific primers (5′-AACCCATTCCACCCCAATATC-3′ and 5′-ATATCTTTGTCGGCCAACTTGTC-3′) as described before (19 (link)). Shrimp β-actin was used as an internal control (primers 5′-CGAGCACGGCATCGTTACTA-3′ and 5′-TTGTAGAAAGTGTGATGCCAGAT CT-3′). Total RNAs were extracted from shrimp hemocytes using RNAprep Pure Cell/Bacteria kit (Tiangen Biotech, China). The cDNA was synthesized with PrimeScript^TM 1st strand cDNA synthesis kit (Takara, Japan). Quantitative real-time PCR reaction mixture contained 5 μl of SYBR® Premix Ex Taq, 0.5 μl of 10 μM forward and reverse primers and 100 ng of cDNA template. The PCR reaction conditions were: 95°C for 1 min, followed by 45 cycles of 30 s at 95°C, 30 s at 52°C, and 30 s at 72°C. For each treatment, quantitative real-time PCR was independently carried out for three times to quantify mRNAs.