Gene Expression Analysis of MERS-CoV Infection

Gene expression analyses were performed as previously described (35 (link)). Briefly, data were collected with the Fluidigm Real-Time PCR Analysis software 4.1.3 (Fluidigm Corporation, USA) and analyzed with the DAG expression software 1.0.5.6 (37 (link)). The relative standard curve method (see Applied Biosystems user bulletin #2) was applied to compare gene expression levels of LN cells cultured in different conditions against those of freshly prepared LN cells, using multiple reference gene normalization (GAPDH, HPRT1 and UbC). Relative expression of IFN-λ1 and IFN-λ3 was calculated according to the 2^-ΔΔCT method (38 (link)), using the same normalizer genes, since expression levels of these genes in control samples were too low to generate standard curves. Relative expression of each gene in a particular sample was expressed in mean fold-change values (Fc) and are shown in Supplementary Table 1.
The unpaired t-test was used to statistically compare the relative expression levels of genes from LN cells exposed to MERS-CoV Qatar15/2015, Egypt/2013 and those of cells cultured in media only. All statistical analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software, USA). Differences were considered significant at p-values < 0.05.