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To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, \u0394Ct method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn\/Vn\u2009+\u20091) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-\u03b2, TNF-\u03b1 and IL-1\u03b2 genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from <jats:italic>F. hepatica<\/jats:italic> and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-\u03b1 and IL-1\u03b2 during chronic stages of infection with <jats:italic>F. hepatica<\/jats:italic> and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in <jats:italic>Fasciola hepatica<\/jats:italic> infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in <jats:italic>F. hepatica<\/jats:italic> vaccinated and infected sheep.<\/jats:p>","DOI":"10.1038\/s41598-018-37672-7","type":"journal-article","created":{"date-parts":[[2019,2,6]],"date-time":"2019-02-06T11:04:47Z","timestamp":1549451087000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica"],"prefix":"10.1038","volume":"9","author":[{"given":"I. 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