2.4 ǀ Expression of cpOAS1 mRNA using quantitative real-time PCR (qPCR)
For qPCR of cpOAS1, primers pairs of OAS1 and Beta-actin (ACTB) genes were used as described in Table 1. ACTB gene was used as an endogenous control. Quantitative real-time PCR was carried out in CFX-96 Real-Time PCR System (BioRad, USA). Primer concentration was optimized using the primer matrix experiments for valid transcript quantification58. All qPCR reactions were performed in duplicates in a volume of 20 μl. The reaction mixture contained 10 pM of each gene-specific primer, 2 μl of cDNA template, 1×SYBRGreen PCR master mix (Sigma, USA) and nuclease-free water to make the total reaction volume of 20 µl. Cycling conditions of PCR were initial denaturation for 10 min at 95°C, followed by 40 cycles of denaturation for 30 s at 95°C; annealing and extension for 60 s at 60°C. The specificity of the amplified product was checked by running a dissociation curve. Mean threshold cycle values (CT) for genes under study were calculated for duplicate samples and relative transcript abundance for target gene expression was calculated using the formula 2-(ΔΔCT)59.