18s rrna taqman assay

Infected Vero cells from single-cycle infections (MOI of three PFU/well, 37°C, described above) were harvested and the cell-associated RNA was collected using the RNeasy Mini Kit (Qiagen). RNA was subjected to strand-specific RT-qPCR to quantify viral negative-sense (genome) and positive-sense (mRNA and antigenome) RNA, as described previously [20 (link)]. Viral RNA was extracted using the RNeasy Mini Kit (Qiagen), and five μg of DNAse-treated RNA was reverse transcribed using SuperScript III First-Strand Synthesis System (Thermofisher) with first-strand primer specific either to genome or to antigenomic/mRNA and linked to an oligonucleotide tag [21 (link)]. Then, each cDNA was amplified in triplicate with a primer containing the oligonucleotide tag, a gene-specific reverse primer, and a probe. Strand-specificity was provided because only cDNAs containing the tagged RT primer sequence would be amplified. QPCR results were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA internal control that had been subjected to RT-qPCR using random first-strand primers and a standard 18S rRNA Taqman assay (Thermofisher). Data were expressed as log₂ fold increase over the Min A four-h time point except for the quantification of wt NS1, NS2, N, P, M, and SH genes in wt RSV-infected cells that were expressed as fold-increase over wt four-h time point.

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.