Quantitative Analysis of Interferon Response Genes

Total RNA was extracted from transfected 293T cells using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions (Gokhale et al, 2020 (link)). RNA concentration and quality were assessed using a NanoDrop spectrophotometer. Complementary DNA (cDNA) was prepared from 500 ng of RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) (Lalani et al, 2015 (link); Gokhale et al, 2020 (link)). Quantitative real‐time PCR of specific genes was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) and primers specific for human IFNB1, MX1, ISG15, OAS1, or GAPDH on a QuantStudio™ 3 Real‐Time PCR System (96‐well, 0.1 ml Block, Applied Biosystems). Post‐amplification melting curve analyses controlled the reaction specificity of each gene. Real‐time PCR data were analyzed using the QuantStudio™ Design and Analysis Software v1.5.2 (Applied Biosystems). Each gene's specific mRNA expression levels were normalized to the housekeeping gene GAPDH. For each target gene, the fold induction of each sample relative to the values of mock‐transfected cells was calculated using the comparative threshold cycle (Ct) method (∆∆Ct) as previously described (Edwards et al, 2014 (link); Lalani et al, 2015 (link)).