Quantifying SARS-CoV-2 RNA Levels in Infected Cells

The cell-associated RNA from infected Vero cells from single-cycle infections was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s recommendations. Then, RNA was subjected to strand-specific RT-qPCR to quantify viral positive-sense (mRNA and antigenome) RNA and negative-sense genomic RNA, as described previously [33 (link),35 (link)] with minor modifications. Briefly, five hundred ng of DNAse-treated RNA were reverse transcribed using SuperScript III First-Strand Synthesis System (Thermofisher) and a primer specific either to antigenomic/mRNA or genomic RNA. Each RT primer was linked to an unrelated oligonucleotide tag [36 (link)] to provide for specific amplification of the RT product at the following PCR step. Then, the cDNA was amplified by qPCR in triplicate with a primer corresponding to the oligonucleotide tag, a gene-specific reverse primer, and a probe. QPCR data were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA internal control that had also been subjected to RT-qPCR in parallel using random first-strand primers and a 18S rRNA Taqman assay (Thermofisher). Data was analyzed using the relative quantification software on the Thermofisher Connect platform and expressed as log₁₀ fold increase over the Min AL 24 h time point.