RNA expression by BMDC
BMDC were cultured at 106 cells/mL in 10 mL of RPMI-1640 complete medium in the presence or absence of 100 ng/mL LPS or 25 µg/mL poly I:C. After 1 and 2 hours, RNA was isolated with TRI-reagent (Sigma) according to the manufacturer's instructions. The expression of Il6, Il12p40, Gmcsf, and Sharpin mRNA was determined by qRT-PCR. Primers and probes were purchased from Applied Biosystems. Reverse transcription was performed at 42°C for 60 minutes with the final denaturation step at 90°C for 5 minutes in 30 µl containing 0.5 µg of total RNA. dNTPs, oligo(dT)15 primer, recombinant RNasin Ribonuclease Inhibitor, and M-MLV Reverse Transcriptase (all from Promega, Madison, WI) were used according to the manufacturer's instruction. Reverse transcription was done in a PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA). qRT-PCR was performed in ABI Prism 7700 Sequence Detection System with TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA) for mouse Actb, IL6, IL12p40, Ifnβ, Gmcsf, and Sharpin according to the manufacturer's protocol. The endogenous standard for normalization of the target gene was β-actin. Relative gene expression was calculated using the 2−ΔΔCt method [56].