Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages

The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. A set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha were validated using QPCR primers and probes which were generated for the equine IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha and 18S genes. Amplification efficiency, intra-assay and inter-assay variation were determined using 10-fold dilutions of plasmid for each gene. Under these conditions the amplification efficiencies of the primers and probes ranged from 99% to 101%. The mean coefficient of variation (CV) across five sets of plasmid DNA for both intra-assay and inter-assay variation was 0.63% (range 0.2% to 1.8%). Amplification efficiency was also determined using 2-fold dilutions of cDNA and under these conditions amplification efficiency ranged from 83% to 95%. The specificity of amplification was confirmed by DNA sequencing of reaction products. The QPCR assays were also evaluated using three sets of cDNA from equine monocyte derived macrophages (EMDM) stimulated for 1 h with lipopolysaccharide (LPS). The general trend was the same for all three samples with IL-1alpha showing the greatest induction and IL-6 the lowest induction. The range of cytokine induction was greater than has previously been reported with values ranging from 12-fold to 30,000-fold. We present a set of QPCR primers and probes that are suitable for quantitation of expression of a set of equine cytokines. The primers and probes have been rigorously analyzed, and we demonstrate that they are specific for the desired genes, have a high amplification efficiency and the assays are highly reproducible.