Intrahepatic mRNA levels of CXCR-3-associated chemokines in individuals infected or uninfected with HCV or HCV/HIV-1.

<p>Chemokine mRNA levels were measured by qPCR assays. Plasmid constructs containing either partial CXCL9, CXCL10, or CXCL11 gene were used to establish standard curves in each qPCR performance for quantitative analysis of CXCL9, CXCL10, or CXCL11 cDNA copy number. Plasmid DNA concentrations were determined using a ND-1000 UV–vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE). The number of construct copies in the plasmid solution was calculated, based on plasmid vector size (pCR2.1-TOPO, 3931 bp, Invitrogen, Carlsbad, CA) and insert size of CXCL9, CXCL10, or CXCL11 gene fragment, respectively. A plasmid-based calibration curve was generated with 10-fold serial dilutions of plasmid containing the CXCL9, CXCL10, or CXCL11 gene fragment sequence; to control pipetting steps, three 10-fold serial dilutions were prepared, and concentrations were checked by real-time PCR. The Y-axis indicates the logarithmic-transformed levels for each chemokine. The lines in each plot show the mean ± standard deviations (SD). The number of subjects per group is indicated in parenthesis. *, <i>p</i><0.05; **, <i>p</i><0.01.