Real-time RT-PCR analysis of ISG15 expression in PBMC. PBMC derived from three distinct healthy subjects numbered #1 (a 46 year-old man), #2 (a 28 year-old man), and #3 (a 42 year-old woman) were incubated for 3 hours or 24 hours in the culture medium with (+) or without (-) inclusion of recombinant human IFNβ, IFNγ, TNFα or IL-1β at a concentration of 50 ng/ml each. cDNA was processed for real-time PCR analysis using specific primers listed in Table 1. The levels of expression of ISG15 are standardized against those of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene detected in identical cDNA samples. The assays were performed in triplicate measurements of the same sample, and the results were expressed as the average with standard error. The panels represent the expression of ISG15 in (a) #1, IFNβ; (b) #2, IFNβ; (c) #3, IFNβ; (d) #1, IFNγ; (e) #1, TNFα; and (f) #1, IL-1β.