Ifnβ1

Total cellular RNA was isolated with Trizol. cDNA was generated with Superscript II and specific primers used for PCR were ISG-15: GCCCACCAAACTGCAGTGCTC and CTGCTGGGGGAGTATGGCCTAAAG; beta actin: GAGGCCCCCCTGAACCCTAAG and GAACCGCTCGTTGCCAATAG; IFN β:GTGGATCCTCCACGCTGCGTTC and TCTCTGCTCGGACCACCATCCA; MDA-5: CCCCGAGCCAGAACTGCAGC and ATCTGCGGCAGGGGAATGGC; RIG-I: CGTTGGGCTGACTGCCTCCG and TGCAGACCCGGCTCTCCTCC; PKR: CGAAAACTGCCGGAACATCC and CTCCACTCCGGTCACGATTTG; ISG1: GCCCACCAAACTGCAGTGCTC and CTTTAGGCCATACTCCCCCAGCAG; ISG56: ACAGCTACCACCTTTACAGC and TTAACGTCACAGAGGTGAGC; IP-10:TCATCCTGCTGGGTCTGAGT and CTGGGTAAAGGGGAGTGATG.
RNA was purified with Qiagen RNeasy MinElute kit for qPCR with Bioline Immomix™, SYBR green and Quantitech 10X primers 18S, IFNβ1, Isg15 or IFIT1 (Qiagen, Manchester, UK). It was performed on an ABI7500 real-time PCR machine and transcript levels were normalized relative to 18S using the ΔΔCt method.

RNA isolation, purification, and reverse-transcription to cDNA were performed as instructed by the manufacturer (Qiagen, Hilden, Germany). Quantification of RNA was done with a NanoDrop ND-1000 spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany). For quantitative real-time PCR (qRT-PCR), Fast SYBR Green Master Mix (Applied Biosystems, Darmstadt, Germany) was used on the 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany). Gene expression analysis was performed with GAPH, Ifnα2, Ifnα4, Ifnα5, Ifnβ1, Usp18, Irf7, Isg15, Mx1, Bst2, Oas1, Irf1, and Bcl2 assays (Qiagen, Hilden, Germany). For analysis, the expression levels of all target genes were adjusted to GAPDH expression levels (ΔCt). Gene expression values were then calculated based on the delta-delta-Ct (ΔΔCt) method relative to the naive controls. Relative quantities (RQs) were determined using the following equation: RQ = 2^−ΔΔCt.