2.7. Quantitative Real-Time PCR Assays

MDBK cells were plated on 24-well cell culture plates at 260,000 cells per well and incubated overnight at 37 °C and 5% CO₂. Once cell monolayers were established, CIGB-325 (15 µM, 30 μM), F20.2 (30 μM), CX-4945 (1.25 μM), or vehicle (PBS) was added for 1 h in serum-free DMEM. Subsequently, 14,000 TCID₅₀ of virus in 200 µL was added to each well (MOI = 0.01). After 1 h of incubation, final volume was completed up to 1 mL, and the appropriate drug’s concentration was maintained for 24 h. Three replicates per condition were used. After incubation time, the culture medium was withdrawn, and the cells were washed with PBS and suspended in 350 µL of Lysis Buffer (with 1% of β-mercaptoethanol, Sigma, St. Louis, MO, USA) for RNA isolation (AllPrep DNA/RNA/miRNA Universal Kit, Qiagen, Valencia, CA, USA), according to the manufacturer protocol. All RNA samples were checked by Nanodrop spectrophotometer to measure concentration (ng/µL) and OD relation (260/280 nm). Quality control parameters were fulfilled by all the samples (100% OD 260/280 nm between 1.7 and 2.2). Complementary (c)DNAs were obtained from 500 ng of total RNAs, using the Transcriptor First Strand cDNA Synthesis Kit package (Roche, Mannheim, Germany), following manufacturer instructions. The qPCR reactions were set up in 20 µL using LightCycler^® 480 SYBR Green I Master 2x (Roche, Mannheim, Germany), 300 nM of oligonucleotides, and 1:10 dilutions of each cDNA, with three replicates per sample. We amplified two genes for normalization GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HMBS (hydroxymethyl-bilane synthase) and the transcript encoding for N protein from BCoV. In parallel, we used a plasmid with the BCoV N protein transcript cloned so that it was amplified from serial dilutions as a standard curve. This standard curve was used for N copy number calculations. All the oligonucleotides were synthetized in the Synthesis Department at CIGB (Table S1). Runs were carried out in the LightCycler^®480II equipment (Roche, Mannheim, Germany) in a 96-well format and SYBR Green Probe II mode with a standard program with 45 cycles. Fold changes of N transcript expression with each treatment were calculated concerning the Virus control using REST 2009 program (v2.0.13, Qiagen GbmH, Munich, Germany) [28], after normalization with GAPDH and HMBS genes, using Ct values and reaction efficiencies per amplicon calculated in LinReg 2009 (v 11.3) [29]. Statistical differences are reported in this program, with a p value-associated significance of p < 0.05 [28]. Additionally, we report a copy number of N transcripts by extrapolation of Ct values into the regression formula from the standard curve (N-BCoV encoding plasmid copy number vs Ct).