Applied cfx96 real time pcr detection system

The number of viral copies in total RNA was measured using methods previously established in our laboratory (7 (link)). Eight genes (IFNα, IFNβ, IFNγ, SOCS3, STAT1, STAT3, MX1, and OASL) and a housekeeping gene (β-actin) were analyzed by qPCR using primers designed with Primer Premier 5 (Table 1). The expression levels of immune-related genes were determined by qPCR using a SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) Kit (TaKaRa) and an Applied CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Amplification was performed in 10 μl reaction volumes containing 0.5 μl of each primer and 1 μl of cDNA. The following thermal cycling conditions were used: initial activation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 58.6°C for 30 s, and a dissociation curve analysis step.

Total RNA was isolated using RNAiso Plus Reagent (TaKaRa) according to the manufacturer’s instructions. Viral RNA levels were determined by RT–qPCR using a One Step PrimeScript RT–PCR kit (TaKaRa) on an Applied CFX96 Real-Time PCR detection system (Bio-Rad). The viral copy number was measured by the following method: standard RNA was diluted in a gradient of EASY Dilution Buffer. According to the sequence of DHAV-1 3D gene (GenBank: JQ316452.1), designed primers (P1: 5′-TGATGAGATATGGCAGGTAGAAGGA-3′; P2: 5′-CACGCAAGTTGATTCACAATAGA-3′) and a probe (FP: FAM-TGTGTTCAGGATCCCCATGTACTACCGTG-TAMRA) were used for this amplification. A gene copies/reaction ranging from 1 × 10¹⁰ to 1 × 10⁵ was used. A regression curve was constructed by plotting the threshold cycle (Ct) values versus the logarithm of the RNA copy number. Kinetic curves and standard curves were obtained with an iCycler IQ Detection System $\left(\mathrm{Y}=-3.274\mathrm{X}+39.955\mathrm{,}{R}^2=0.998\right)$ . The viral copy number was obtained by substituting Ct values from samples into the equation representing the standard curve.

Total RNA was isolated using RNAiso Plus Reagent (TaKaRa) according to the manufacturer’s instructions. The number of viral copies in total RNA was measured using methods previously established in our laboratory (Hu et al., 2016 (link)). Three genes (PKE, PERK, and GCN2) and a housekeeping gene (β-actin) were analyzed by qPCR using primers designed with Primer Premier 5 (Table 1). The expression levels of four genes were determined by qPCR using a SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) Kit (TaKaRa) and an Applied CFX96 Real-Time PCR Detection System (Bio-Rad). Amplification was performed in 10 μl reaction volumes containing 0.5 μl of each primer and 1 μl of cDNA. The following thermal cycling conditions were used: initial activation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 56.9°C for 30 s, and a dissociation curve analysis step.