RNA Extraction and RT-qPCR Protocol

Total cellular RNA was extracted using TRI reagent (MRC) or a GenElute total RNA miniprep kit (Sigma) according to the manufacturer’s protocol. RNA samples were treated with RNase-free DNase (Qiagen or Sigma) prior to cDNA synthesis. Total RNA(1 μg) was reverse transcribed by using iScript cDNA synthesis kit (Bio-Rad). RT-qPCR was performed using iQ SYBR green supermix (Bio-Rad) in an Applied Biosystems StepOnePlus real-time PCR machine. Gene expression was calculated using the 2^-△△CT method[61 (link)], normalized to actin. QuantiTect primers against ADAP2, IFI44L, DENV, VSV, were purchased from Sigma. Primer sequences were as follows: ADAP2 (5’-AAGCTGTCATCAGCATTAAG-3’ and 5’-ACTATCTCCTTCCCACTTTC-3’); IFI44L (5’-ACTAAAGTGGATGATTGCAG-3’ and 5’-TGCAGAGAGGATGAGAATATC-3’); DENV (5’-AGTTGTTAGTCTACGTGGACCGA-3’ and 5’-CGCGTTTCAGCATATTGAAAG-3’). Actin, VSV, CVB, ISG56, ISG60 and SeV primer sequences have been described [36 (link), 62 (link)].