Quantitative PCR

RNA was prepared from cell lysates and homogenized murine lungs by TRI Reagent or acid-Phenol:Cholorform:IAA 125:24:1 pH 4.5 extraction (Ambion), respectively. cDNA was prepared using high-capacity cDNA reverse transcriptase kit (Applied Biosystems), genomic DNA removed by DNA-free™, and qPCR performed using GoTaq® master mix (Promega, Madison, US) and primer-probe sets from Applied Biosystems. Cell culture samples were quantified against a plasmid standard curve containing known copy numbers of target genes; murine samples were analyzed using the relative quantification (2^−ΔΔCt) method to 18sRNA.

Probes used were GAPDH Hs00182082_m1; IFNβ Hs01077958_s1; murine CXCL10 Mm00445235_m1; murine IFNβ Mm00439552_s1; murine IL-6 Mm00446190_m1; murine Rn18s Rn45s Mm04277571_s1;

RV1B F 5′-GTGAAGAGCCSCRTGTGCT-3′,

R 5′-GCTSCAGGGTTAAGGTTAGCC-3′,

probe 5′-[6FAM]TGAGTCCTCCGGCCCCTGAATG[TAM]-3′;

X31 F 5′-CATCCTGTTGTATATGAGGCCCAT-3′,

R 5′-GGACTGCAGCGTAGACGCTT-3′,

probe 5′-[6FAM]CTCAGTTATTCTGCTGGTGCACTTGCCA[TAM]-3′.