Quantitative RT-PCR Analysis of Type I Interferon Pathway

Total RNA from whole blood cells was isolated by way of the PAXgene Blood RNA kit (Qiagen) according to the manufacturer’s recommendations. Total RNA was obtained from skin samples, cDNA was synthesized, and real-time polymerase chain reactions (quantitative RT-PCR) were performed as previously described (35 (link)). Oligonucleotide sequences used in the RT–qPCR assays are displayed in Supplementary Table 6. The reactions were incubated in the StepOnePlus^® real-time PCR equipment (Applied Biosystems, United States) as described (36 (link)). For each sample, the cycle- threshold (CT) means of the genes of interest (IFNB, IFNAR1, IFI16, TBK1, EIF2AK2, and MX1) were normalized by the CT mean of the reference gene RPL13a (ThermoFisher Scientific, United States). The relative gene expression analysis was performed utilizing the 2^–ΔCT method for each target gene (37 (link)).