Quantifying RSV Infection in Mouse Lungs

Lungs were homogenized using a TissueLyser LT (Qiagen) and total RNA was extracted from the lung tissue supernatant using RNeasy Mini kit (Qiagen). RNA concentration was determined by NanoDrop (Thermo Scientific). 2 µg of RNA was converted to cDNA using High Capacity RNA-to-cDNA kit (Applied Biosystems). RT-qPCR was performed with Quantitect Probe PCR Master Mix (Qiagen). RSV L gene mRNA was quantified as previously described^{12 (link)}. For exact quantification, copy numbers were calculated from a plasmid DNA standard curve and normalized to Gapdh (encoding glyceraldehyde-3-phosphate dehydrogenase). For mRNA analysis of Gapdh, Cxcl1, Cxcl2, Cxcl12, Il1a, Il1b, Il33, Areg, Ptgs2, Muc5ac gene specific primers and probes were used (all Applied Biosystems). RT-qPCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems). To quantify relative mRNA expression the mean ΔCT was calculated for each target gene relative to Gapdh and expressed as 2^−ΔCT. Analysis was performed using 7500 Fast System SDS Software (Applied Biosystems).
Live virus was quantified in the lungs by immunoplaque assay as previously described^{12 (link),36 (link)}.