Quantification of Lung Gene Expression

Lung tissue was homogenized using a TissueLyser LT (Qiagen), and total RNA was extracted using RNeasy Mini kit including DNA removal (Qiagen) according to the manufacturer’s instructions. 1-2 μg of RNA was reverse-transcribed using the High Capacity RNA-to-cDNA kit according to the manufacturer’s instructions (Applied Biosystems). qPCR was performed to quantify lung RNA levels. To quantify Ifng, Tnfa and L gene primers and FAM-TAMRA probes previously described were used^{22 (link)}. The absolute number of gene copies was calculated using a plasmid DNA standard curve and the results were normalized to levels of Gapdh (Applied Biosystems). For relative quantification, the expression of Adora2a, Adora2b, Adora3 Cd200, Cd200r1, Cxcl1, Cxcl10, Ifna5, Il1b, Il6, Il28b Sftpa1, Sftpc, Sftpd, Tlr7 (all from Applied Biosystems run with the mastermix QuantiTech Probe PCR kit (Qiagen)) and influenza NP gene (primers from ref^{59 (link)} run with SYBR green master mix (Thermo Fisher)) were expressed relatively to the expression of Gapdh. First, the ΔCT (Ct = cycle threshold) between the target gene and the Gapdh for each sample was calculated. Then the expression was calculated as 2^-ΔCt. Analysis was performed using 7500 Fast System SDS software (Applied Biosystems).