Quantitation of cytokine mRNAs by qPCR.

Total RNA was isolated from spleens by use of a SimplyRNA kit (Promega) according to the manufacturer's instructions. One microgram of RNA was used to synthesize cDNA by use of a high-capacity cDNA reverse transcription kit (Thermo Scientific) according to the manufacturer's instructions. Transcripts for cytokine genes (IL-4, IL-10, IL-12, IFN-β, TNF-α, and IFN-γ) were quantified by qPCR with SYBR green master mix (Roche), using an ABI Prism 5700 sequence detection system (Applied Biosystems). The oligonucleotide primers used for qPCR are shown in Table 1. Primers for IL-4 (38), IL-12 p40 (39), IL-12 p35 (40), IFN-γ (41), TNF-α (42), and 18S rRNA (43) were previously published. Primers for IFN-β were validated by IDT. Primers for IL-10 were designed by use of the NCBI Primer Blast tool and then validated. Relative fold changes were determined using the threshold cycle (2^−ΔΔCT) method, with the 18S rRNA gene used as a reference gene for normalization, as previously described (44). Briefly, amounts of cytokine mRNA were normalized to the amount of internal 18S rRNA. The fold change of cytokine mRNA for each infected mouse was calculated by dividing the amount of cytokine mRNA in infected mice by that in uninfected mice.

Primers used for quantitative RT-PCR