Quantification of POWV RNA Levels

Total intracellular RNA was isolated using the RaPure Total RNA Kit (Magen). To analyze the POWV RNA levels in replicon cells, quantitative RT-PCR was performed. In brief, 1 µg total RNA was reverse transcribed using the ReverTra Ace qPCR RT Kit (TOYOBO, FSQ-101) to produce cDNA with random primers. Reactions of qPCR were carried out using the 2× RealStar Green Power Mixture (Genstar, A311) according to the manufacturer’s instructions. The qPCR primers for viral RNA were as follows—POWV: THU-5193 (5′-CGC CCT CAA CAC CAT CAC AAA C-3′) and THU-5194 (5′-TCA ACT CCG TGC TCC TTC AAC C-3′). The sequences of the qPCR primers for GAPDH were described previously (45 (link)). Relative expression levels of the target genes were calculated using the comparative cycle threshold method. All data were normalized relative to GAPDH.