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.