Reference gene analysis and its use for kinase expression profiling in Fasciola hepatica

Abstract

AbstractThe liver flukeFasciola hepaticacauses fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR inFasciolastudies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis ofin vitro-cultured juveniles were Fhtbcdand proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping genegapdhwhich is used in mostFasciolastudies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for futureF.hepaticaresearch, such as studies on drug targets or parasite development.