Fig. 1.

Development of TaqMan assay to detect expression of ferret immune mediators and housekeeping genes using plasmid DNA standards. (A) The specificity of all primer/probe sets was tested against all DNA plasmid standards (1 pg) by TaqMan real time PCR and assessed by gel electrophoresis. An example is shown. (B) The efficiency of each reaction was determined using a 10-fold dilution standard curve. Cytokine and chemokine (black circles) and housekeeping (white circles) genes are shown. The mean efficiency for all genes is indicated by the horizontal line. The reaction efficiency (E) is indicated with the corresponding % efficiency, with the ideal value ‘E = 2’ and the acceptable range (1.9–2.1), indicated. (C) One or two primer/probe sets were combined in a real time PCR reaction and assayed against each of the single target genes. (D) One primer/probe set was assayed against the target gene in a pool (four to seven plasmids) or alone. (E) One or two primer/probe sets were combined in a real time PCR reaction and assayed against the target gene in a paired pool. (C–E) All samples were run in triplicate, with mean and standard deviation indicated. *p < 0.05.