Cell culture or lung RNA samples were collected and incubated in RNAlater reagent (Ambion). To extract total RNA, lungs were homogenized in 2 mL of RLT lysis buffer (Qiagen) supplemented with 1% β-mercaptoethanol using the GentleMACS Dissociator system (Miltenyi Biotec) following the protocol proposed by the manufacturer. RNA was purified from supernatants using the rNeasy minikit reagent (Qiagen), as previously described. To generate cDNAs from the purified RNAs, a reverse transcription (RT) reaction was carried out using the High Capacity cDNA RT kit reagent (Applied Biosystems). To verify the sequence of the mutations introduced into the viral genome, 4 μL of the cDNA generated in the RT reaction was used as a template in a PCR using the enzyme Taq polymerase (Invitrogen). Cellular mRNA expression was analyzed by quantitative reverse transcription-quantitative PCR (RT-qPCR), using TaqMan technology with commercial probes (ThermoFisher Scientific) (Table 2 ). In all cases, the reaction was performed with the FastStart Universal Probe Master reagent (Rox) (Roche). qPCRs were performed in a 7500 Real Time PCR System (ThermoFisher Scientific). qPCR data were analyzed using the 7500 software v2.0.6 (ThermoFisher Scientific). All experiments met the recommendations for analysis of gene expression by RT-qPCR (MIQE) (79 (link)). Each result is the average of three independent experiments in which each sample was tested in triplicate. The relative quantification of gene expression was performed from the mean values of CT (cycle in which the amplification curve cuts at the threshold level using the 2−ΔΔCT method) (80 (link)). The level of 18S rRNA was used as an endogenous control to normalize cellular mRNA levels in mouse lungs, while hydroxymethylbilane synthase (HMBS) was used to normalize cellular mRNA levels in cell cultures.
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