Sybr greenimaster

In order to determine the replication of HIRRV in mIgM⁺ B lymphocytes, 1 μg total RNA extracted from mIgM⁺ B lymphocytes was reversely transcribed into cDNA in a 20 μL reaction. Then, 2 μL cDNA was used as the templates, and a pair of specific primers (F:5’-CTTCCTGATTGTGATGTCTGCG-3’and R:5’-CAACGATACTCC TGTGATTCCG-3’) were designed for PCR amplification of the fragment of HIRRV glycoprotein (G) gene. Each sample was run in triplicate, and the non-infected samples were used as the negative control. After amplification, melting curve analysis was performed to ensure no nonspecific amplification. Finally, viral copy numbers were determined by extrapolating Ct values from the standard curve which was established previously (4 (link)). The data was expressed as mean log₁₀ copies/0.1 μg RNA.
Specific primers of immune-related genes were designed according the sequencing results with Primer Premier 5, 18S rRNA of flounder was used as the internal reference to normalize the expression level. The qRT-PCR was performed using SYBR GreenIMaster (ABM) in LightCycler^® 480 II Real Time System (Roche), and the expression levels of selected genes were analyzed by the 2^−△△Ct method, the primers used in this part were listed in Table 1.

Based on the identification result of proteomic and phosphoproteomic analysis, eight antiviral-related mRNA, including ISG15, IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44 and MX, were further screened, and their temporal expression profiles were examined by reverse transcription quantitative real-time PCR (RT-qPCR). The specific primers for above eight genes were designed using Primer Premier software (Version 5.0) and listed in Table 1, we chose 18s rRNA as the internal reference gene. The qRT-PCR was performed on LightCycler^® 480 II Real Time System (Roche) using SYBR GreenIMaster (ABM), and the expression levels of selected genes were analyzed by the 2^−ΔΔCt method [34 (link)].