title: Development of a multiplex quantitative PCR assay for the analysis of human cytokine gene expression in influenza A virus-infected cells.
abstract: 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 20fmol 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.
authors.affiliation: Research Institute of Influenza, Saint-Petersburg, Russian Federation. Electronic address: biomalinka@gmail.com.
authors.forename: Marina A
authors.initials: MA
authors.lastname: Plotnikova
authors.affiliation: Research Institute of Influenza, Saint-Petersburg, Russian Federation.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Klotchenko
authors.affiliation: Research Institute of Influenza, Saint-Petersburg, Russian Federation; Peter the Great St. Petersburg Polytechnic University, Russian Federation.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Vasin
mesh_terms: D002460:Cell Line; D016207:Cytokines / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000276:immunology* / Q000502:physiology; D016753:Interleukin-10 / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology; D015847:Interleukin-4 / Q000235:genetics / Q000276:immunology; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D014779:Virus Replication / Q000235:genetics
chemical_list: D016207:Cytokines; C508609:IL10 protein, human; D007376:Interleukin-2; D016753:Interleukin-10; D015847:Interleukin-4
keywords: Cytokines; Influenza virus; Multiplex quantitative real-time PCR