Evagreen 2 qpcr master mix

For cDNA synthesis, 1000 ng of intact RNA was reversed to cDNA with a reverse transcription kit (YEASEN, Shanghai, China) with the concentration detected. The synthesized cDNA was diluted to 200 ng/μL as a template for RT-qPCR. To detect carbohydrate metabolism, the expression of genes related to glycogen synthesis (ugp2b, gys2), glycogen degradation (pygl), gluconeogenesis (pck1, pcxb), glycolysis (gck), TCA cycle pathway (idh), and pentose phosphate pathway (g6pd) were evaluated. The expression of genes related to lipid synthesis (fasn, acaca, aclyb) and decomposition (acadl, acaa1, lpl) were determined to illustrate the influence on lipid metabolism, and the expression of genes related to the urea cycle (gs, cps3, otc, ass, asl, and arg1) was also detected. The qPCR was performed in Jena qTOWER3G system using the real-time quantitative PCR detection kit EvaGreen 2 × qPCR Master mix (YEASEN, Shanghai, China): pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 10 s; annealing and extension at 60 °C for 30 s; and PCR reaction step running 40 cycles. After RT-qPCR, melting curves were analyzed to ensure the specificity of the reaction. Using 18s as the internal reference gene, the relative quantitative data analysis was performed by the 2^−ΔΔCt method. RT-qPCR data were analyzed using GraphPad Prism 6. The primers used in the present study are shown in Table 1.

To study the mRNA expression pattern of ICAM-1 in different tissues, healthy rainbow trout were anesthetized with MS-222, and then tissues including the head kidney, spleen, gills, muscle, gut, stomach, skin, liver, nose, buccal mucosa, pharynx, and eye were collected. Total RNA was extracted from the tissues and then subjected to reverse transcription with the SuperScript first-strand synthesis system for RT-qPCR (Yeasen, China). The expression levels of elongation factor 1α (EF-1α, housekeeping gene) and ICAM-1 were detected by RT-qPCR using the EvaGreen 2× qPCR Master mix (Yeasen, China) by a 7500 qPCR system (Applied Biosystems), 10 μl qPCR reaction mixture containing 5 μl EvaGreen 2× qPCR Master mix, 1 μl of the cDNA, 0.15 μl each of forward and reverse primer, and 3.7 μl H₂O was performed for amplifications. The qPCR conditions were: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s and at 58°C for 30 s. Primer sequences can be found in Supplementary Table 1.

Total RNA was extracted from various tissues using a TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. The concentration of extracted RNA was determined by spectrophotometry (Nanodrop ND1000, LabTech), and the integrity of the RNA was determined by 1% agarose gel electrophoresis (Agilent Bioanalyser, 2100). To normalize gene expression levels for each sample, equivalent amounts of total RNA (1,000 ng) were used for cDNA synthesis with the SuperScript first-strand synthesis system in a 20-μl reaction volume. The synthesized cDNA was diluted three times and then used as a template for qRT-PCR analysis. The qRT-PCR was performed by the qTOWER3G PCR system (Analytik Jena AG, Germany) using the EvaGreen 2 × qPCR Master mix (YEASEN, China) as the following conditions: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s and at 58°C for 30 s. The qRT-PCR products of the SVCV were verified by 2% agarose gel electrophoresis. Ct values determined for each sample were normalized against the values of 40S, and the change in transcription of genes was calculated as relative fold expression by the methods of 2^-ΔΔCt. Primers used for qRT-PCR are listed in Supplementary Table 1.