Quantitative Real-Time PCR Analysis

Total RNA was prepared using the RNAqueous-96 Isolation Kit (Invitrogen-Thermo Fisher, Carlsbad, CA, USA) following the manufacturer’s instructions. Total RNA (1 µg) was subjected to reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen, Toronto, ON, Canada). Quantitative PCR were performed using either Fast start SYBR Green Kit (Roche, Indianapolis, IN, USA) for MX1, IDO, APOBEC3G, CXCL10, NOD2, PKR, IRF1, IFIT1 and IL8 or TaqMan Gene Expression Assays (Life Technologies-Thermo Fisher) for DUOX2, IFI27, SERPINB2, IL33, CCL20, ISG20. Sequences of oligonucleotides and probes used in PCR reactions are described in Supplemental Table S4. Data collection was performed on a Rotor-Gene 3000 Real Time Thermal Cycler (Corbett Research, Mortlake, Australia). Gene inductions were normalized over S9 levels, measured using Fast start SYBR Green Kit or TaqMan probe as necessary. Fold induction of genes was determined using the ΔΔCt method [19 (link)]. All qRT-PCR data are presented as the mean ± standard error of the mean (SEM).