Quantitative Real-Time PCR for Viral RNA and Host Gene Expression

Total RNA from cell samples in each well was separately extracted using the RNA Extraction Kit (Biotek, RP4002). Purity and concentration of the RNA samples were measured using NanoDrop (Thermo Fisher). Total RNA was used to synthesize cDNA with HiScript^® 1st Strand cDNA Synthesis Kit (Vazyme, R211-02) according to the manufacture’s protocol.
For qPCR, the HiScript One Step qRT-PCR Probe Kit (Vazyme, Q222-01) was used to detect the viral RNA copies, and SYBR Green Kit was used to analyze expression of cellular genes on a Mx3005P Real-Time PCR system (Agilent, United States). Gene-specific primers are listed in Table 1. The cycling parameters included initial denaturation at 95°C for 30 s, followed by 45 cycles (5 s at 95°C and 40 s at 60°C), with a final cooling at 42°C for 30 s. The viral RNA copies were calculated as described (Zhou et al., 2019 (link)). The qPCR parameters for quantification of relative expression of cellular genes were: 95°C for 5 min for initial denaturation; 40 cycles of 95°C for 10 s, 60°C for 30 s, followed by a dissociation curve segment (95°C, 1 min; 60°C, 30 s; 95°C, 30 s). The relative expression of the host cell genes was normalized to GAPDH.