Hyperscript first strand synthesis kit

EPC cells were cultured in 6-well plates (1.5 × 10⁶ cells/well) at 15 °C and were infected with either the recombinant VHSVs, rVHSV-wild, or rVHSV-dNV at MOI 0.1. At 12-, 24-, and 48-h post-infection, the total RNA was extracted from the infected EPC cells (rVHSV-wild or rVHSV-dNV) using a Hybrid-R Kit (Gene All, Korea), and 1 μg of total RNA was used to synthesize cDNA using a HyperScript First Strand Synthesis Kit with oligo (dT) primer or random primer (Gene All) according to the manufacturer’s instructions. We performed real-time RT-PCR using a Light Cycler 480 (Roche) and constructed each vector to contain a G-NV region or an NV gene for absolute quantification. cDNA synthesized with a random primer was used to quantify each virus genome, and cDNA synthesized with oligo(dT) was used as a template to amplify the NV gene transcript alone, whose primers are in Table 1. We ran the PCR reactions in a 20 μL volume using 2× SYBR Green Premix (Enzynomics) with 5 μL of cDNA and 5 pM of each primer with the thermal cycling conditions: 1 cycle of 15 min at 95 °C (pre-incubation), followed by 40 cycles of 10 s at 95 °C, 10 s at 60 °C, and 20 s at 72 °C.

Total cellular RNA was extracted from cells using RNX-Plus reagent (Cinagen Co., Tehran, Iran) according to the manufacturer’s instruction.
The concentration of the purified RNA was determined by measuring absorbance at 260 and 280 nm. The quantity of messenger RNA (mRNA) was measured by means of Nanodrop ND-1000 (Nanodrop Technologies, DE, USA). Total mRNA was first normalized in concentration (up to 2 μg/ml) and applied to synthesize complementary DNA (cDNA) using HyperScript Reverse Transcriptase following the manufacturer’s protocol (HyperScript First-Strand Synthesis Kit, GeneAll, South Korea). The reaction mixture for real-time PCR was prepared by combining 12.5 μl RealQ Plus 2x Master Mix Green (AMPLICON, Denmark), 1 μl of each primer (10 μM), PCR-grade distilled water and template cDNA (average 100 ng/μl) in a total volume 25 μl. The PCR primer sequence for genes was listed in Supplementary Table 2. The annealing temperature for reactions was 62 °C. Each sample was amplified in triplicate experiments. The relative expression was quantified using 2^{−Δct (cycle threshold)} formula using HGPRT (hypoxanthine-guanine phosphoribosyltransferase) transcription levels as a reference gene^{44 (link)}. Fold change of expression was also measured using 2^−ΔΔct formula^{44 (link)}.

Changes in the mRNA level of NF-kB, IL-1β, and Casp9 were measured. The total RNA was isolated from the liver tissues using the High Pure RNA Kit according to the manufacturer’s protocol (Roche, Germany). The RNA concentration and quality were determined spectrophotometrically at 260 nm and by the A260/A280 ratio, respectively. Subsequently, 0.1 μg of RNA from each sample was subjected to reverse transcription using the HyperScript™ First-strand synthesis kit (GeneAll, Seoul, Republic of Korea). A real-time polymerase chain reaction (RT-PCR) was performed with a Rotor-Gene Q (Qiagen) using RealQ Plus 2× Master Mix Green (Ampliqon, Odense, Denmark) [35 (link)]. The thermal cycling conditions involved an initial activation step for 15 min at 95 °C followed by 40 cycles, including a denaturation step for 20 s at 95 °C and a combined annealing/extension step for 60 s at 60 °C. Melting curves were analyzed to validate a single PCR product of each primer. The fold change in expression of each target mRNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined using the 2^–ΔΔct method. Primer sequences are stated in Table 1.