Abstract
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-β, TNF-α and IL-1β 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-1β 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.
Highlights
Fasciola hepatica is the causative agent of fasciolosis in temperate climates, infecting a wide range of hosts — ruminants — and resulting in estimated economic losses of US $3,200 million per annum worldwide to the agricultural sector[1]
Changes in expression patterns of FoxP3, IL-10, TGF-β, TNF-α and IL-1β in the hepatic lymph nodes (HLN) of vaccinated sheep infected with F. hepatica, and TNF-α and IL-1β in the liver of sheep unvaccinated and infected with F. hepatica were investigated using the selected reference genes, PGK1, HSP90AA1 and GYPC
In order to identify a set of reference genes suitable for use under different experimental conditions in two key immunological tissues (HLN and liver), we performed stability studies on a select group of putative reference genes
Summary
Fasciola hepatica is the causative agent of fasciolosis in temperate climates, infecting a wide range of hosts — ruminants — and resulting in estimated economic losses of US $3,200 million per annum worldwide to the agricultural sector[1]. Quantitative real-time polymerase chain reaction (qRT-PCR) is efficient, sensitive and probably the most reliable approach for accurate quantification of gene expression from different sources and samples. This methodology, being relatively simple and cost-effective, is considered the gold standard for measuring the copy number of specific targets[12]. The present study sought to determine the expression stability of 10 commonly used reference genes in order to identify those most suitable for qRT-PCR normalisation in experiments performed on liver and lymph node tissues of F. hepatica-infected sheep. Changes in expression patterns of FoxP3, IL-10, TGF-β, TNF-α and IL-1β in the hepatic lymph nodes (HLN) of vaccinated sheep infected with F. hepatica, and TNF-α and IL-1β in the liver of sheep unvaccinated and infected with F. hepatica were investigated using the selected reference genes, PGK1, HSP90AA1 and GYPC
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