Quantifying RSV and Inflammatory Genes in Lungs

RNA extraction from lung tissue was performed using Trizol (Invitrogen) according to manufacturer’s instructions. One microgram of RNA was reverse-transcribed using a High Capacity RNA-to-cDNA kit according to manufacturer’s instructions (Applied Biosystems). To quantify mRNA levels in lung tissue quantitative RT-PCR was performed. RT-PCR reactions for Areg (Amphiregulin), Il1a, Il1b, Cxcl9, Cxcl10 (Applied Biosystems) and RSV L gene23 (link) were performed using the Quantitect Probe PCR Master Mix (Qiagen) and the 7500 Fast Real-time PCR System (Applied Biosystems). For absolute quantification of RSV L gene, the exact number of copies of the gene was calculated using a plasmid DNA standard curve and then normalised to levels of Gapdh, a housekeeping gene (Applied Biosystems). For relative quantification, the expression of Areg, Il1a, Il1b, Cxcl9 and Cxcl10 was expressed relatively to the expression of Gapdh. First, the ΔCt (Ct = cycle threshold) between the target gene and Gapdh for each sample was calculated, following the calculation of 2^−ΔCt. Analysis was performed using 7500 Fast System SDS Software (Applied Biosystems).