Sensifast sybr no rox mix

To investigate the effect of cordycepin on DENV replication, Vero cells were seeded at 1.5 × 105 cells per well in a 12-well plate for 24 h before addition of DENV2 for 2 h at 37 °C with 5% CO₂. The unbound viruses were removed and cordycepin at concentrations of 50 and 100 µM was added. The cells were harvested at 4, 8, and 12 h, and the total RNA was extracted using TRIzol™ Reagent (Invitrogen). The RNA (1.0 µg) was converted into cDNA using a cDNA Synthesis Kit (Toyobo Life Science, Osaka, Japan). The PCR mixture contained 0.5 µg of cDNA, DENV E-specific primers [10 µM of D2R (5′-CCGGCTCTACTCCTATGATG-3′), and 10 µM of D2L (5′-ATCCAGATGTCATCAGGAAAC-3′)], and 2 × SensiFAST SYBR No-ROX Mix (Meridian Bioscience, Cincinnati, OH, USA). RT-PCR was then performed on an iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The data were normalized with the housekeeping gene GAPDH and analyzed for relative value to that of non-treated control (set as 1).

Stage-1 quantitative PCRs (qPCRs) for both the ITS-2 rDNA and isotype-1 β-tubulin nemabiome assays were performed on all gDNA lysates in-house (Francis et al., 2020 (link)). Illumina adapter primers were previously described and validated (Avramenko et al., 2015 (link); Avramenko et al., 2019 (link)). For both assays, equal concentrations of the forward and reverse primers were mixed to create single forward and reverse primer mixes with final concentrations of 10 μM. qPCRs were performed at a final volume of 20 μL, containing SensiFAST™SYBR® No-ROX mix (Meridian Bioscience, Australia), forward and reverse primer mixes and 2 μL of template DNA. Both qPCR assays were run on a CFX96 Real-Time PCR Detection System (BioRad, Australia) under the following 3-step conditions: initial denaturation at 95°C for 3 min, followed by 25–35* cycles of 95°C for 5s, 60°C for 15s, 72°C for 15s and a final melt curve analysis (*cycles were carefully selected to ensure all reactions were still in their exponential phase and had not plateaued). All qPCR runs included a no template control (ddH₂O) and the first 10 reactions were run on a 1% agarose gel stained with 0.1% GelRed (Biotium, USA) and visualized under a UV transilluminator to ensure amplicons were the required size.

MOVAS cells were incubated for 24–48 h with or without 9 mg/dL UA. Total RNA was isolated using the Tri-Reagent (Zymo Research, Aurogene, Rome, Italy). A total of 1 µg RNA was used for cDNA synthesis with SCRIPT RT Supermix (BioRad Laboratories, Segrate, Italy). PCR amplification was carried out in a total volume of 10 µL, containing 1 µL cDNA solution, 5 µL SensiFAST SYBR no ROX mix (Meridian Bioscience, Aurogene, Rome, Italy), 0.08 µL of each primer (Tib Molbiol, Genoa, Italy) and 3.84 µL of nuclease-free water. β-actin was quantified, and used for the normalization of expression values of the other genes.
Assays were run in triplicate on MasterCycler realplex PCR system (Eppendorf, Hamburg, Germany). The primer sequences are reported in Table 1.