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Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica.
Pacheco, I L; Abril, N; Zafra, R; Morales-Prieto, N; Hernández, V Molina; Ruiz, M T; Perez-Caballero, R; Martínez-Moreno, A; Pérez, J.
Afiliação
  • Pacheco IL; Department of Anatomy and Comparative Pathology, University of Córdoba, Córdoba, Spain.
  • Abril N; Department of Biochemistry and Molecular Biology, University of Córdoba, Córdoba, Spain.
  • Zafra R; Department of Animal Health, Parasitology section, University of Córdoba, Córdoba, Spain.
  • Morales-Prieto N; Department of Biochemistry and Molecular Biology, University of Córdoba, Córdoba, Spain.
  • Hernández VM; Department of Anatomy and Comparative Pathology, University of Córdoba, Córdoba, Spain.
  • Ruiz MT; Department of Anatomy and Comparative Pathology, University of Córdoba, Córdoba, Spain.
  • Perez-Caballero R; Department of Animal Health, Parasitology section, University of Córdoba, Córdoba, Spain.
  • Martínez-Moreno A; Department of Animal Health, Parasitology section, University of Córdoba, Córdoba, Spain.
  • Pérez J; Department of Anatomy and Comparative Pathology, University of Córdoba, Córdoba, Spain. an1pearj@uco.es.
Sci Rep ; 9(1): 1485, 2019 02 06.
Article em En | MEDLINE | ID: mdl-30728395
The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in hepatic lymph nodes (HLN) and livers from sheep infected with Fasciola hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) 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-ß, TNF-α and IL-1ß genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1ß during chronic stages of infection with F. hepatica 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 Fasciola hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. hepatica vaccinated and infected sheep.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Contexto em Saúde: 3_ND Problema de saúde: 3_neglected_diseases / 3_zoonosis Assunto principal: Ovinos / Fasciola hepatica / Fasciolíase Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Animals Idioma: En Revista: Sci Rep Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Espanha

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Contexto em Saúde: 3_ND Problema de saúde: 3_neglected_diseases / 3_zoonosis Assunto principal: Ovinos / Fasciola hepatica / Fasciolíase Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Animals Idioma: En Revista: Sci Rep Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Espanha
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