Quantitative real-time polymerase chain reaction (PCR) primers and probes were obtained (TaqMan; Applied Biosystems; Foster City, California). For detection of MxA, ISG15, IFN-α, IFN-β, and IFNAR1, predeveloped TaqMan gene expression assay reagents were available. As the housekeeping endogenous control for sample normalization, 18S ribosomal RNA (rRNA) was used. The internal fluorescent TaqMan probe and 18S rRNA specific primers were designed as follows: 5′-CAT-TCT-TGG-CAA-ATG-CTT-TCG-3′, 5′-CGC-CGC-TAG-AGG-TGA-AAT-TC-3′ (TaqMan probe, Applied Biosystems) and VIC-ACC-GGC-GCA-AGA-CGG-ACC-AGA-TAMRA (18S probe, Invitrogen). The messenger RNA (mRNA) levels of the genes MxA, ISG15, IFN-α, IFN-β, and IFNAR1 and of 18S rRNA were measured in adherent and nonadherent PBMC samples from our patient with dermatomyositis and from 2 controls (ABI Prism 7700 Sequence Detection System, Applied Biosystems). The PCR was performed (TaqMan Universal PCR MasterMix, Applied Biosystems) using the following conditions: 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Dilution experiments were performed to ensure similar efficiency of the PCR. The relative expression levels of MxA, ISG15, IFN-α, IFN-β, and IFNAR1 were calculated according to the 2−ΔΔCt method.17- 19 Briefly, the cycle threshold (Ct) values of triplicate real-time PCR reactions were averaged for each gene in each complementary DNA sample. For each sample assayed, the mean Ct value for the gene of interest was subtracted from the mean Ct value of 18S rRNA to obtain the ΔCt value. The ΔΔCt value was calculated by subtracting the mean ΔCt calibrator values from the ΔCt sample values. The relative quantification was then calculated by 2−ΔΔCt. The mRNA quantity for the calibrator is expressed as the 1× sample, and all other quantities are expressed as a number of fold differences relative to the calibrator. The mean ΔCt of the normal adherent sample without IFN-β was used as the calibrator for the adherent samples, and likewise the mean ΔCt of the normal nonadherent sample without IFN-β was used as the calibrator for the nonadherent samples. The standard deviation values for ΔΔCt were added to, or subtracted from, the ΔΔCt to generate the upper and lower ΔΔCt standard deviation boundaries. To generate error bars, these ΔΔCt values were then transformed (2−(ΔΔCt±s), where s is the standard deviation of the ΔΔCt value).