Development of a multiplex quantitative PCR assay for the analysis of human cytokine gene expression in influenza A virus-infected cells

Cytokines are global mediators of cellular communications that are involved in broad array of biological processes, including the immunological and inflammatory mechanisms of virus-host interactions. Measuring the gene expression of simultaneously expressed cytokines is necessary for understanding the pathogenesis of many viral infections, including influenza. We developed a multiplex quantitative real-time PCR (qPCR) method for the detection of the following human cytokines: IL-1B, IL-2, IL-4, IL-6, IL-10, IL-12B, IL-18, IFN-γ and TNF. The assay consisted of three sets of multiple qPCRs; in each qPCR, three target cytokines and reference GAPDH genes were amplified. The assay provided a precise and sensitive quantification of cytokine gene expression with a 20 fmol limit of detection and a 1.5% coefficient of variation. This method was successfully applied to cytokine profiling in epithelial A549 cells that were infected with A/California/07/09 (H1N1pdm2009) virus.