Discussion

In Fasciola hepatica research, comparative gene expression analysis of the different life-stages is of major interest. This includes expression analysis of potential drug target genes: a new active compound should ideally target all life stages within the mammalian host, from NEJ and immature to adult flukes. Other research approaches include the in vitro-culture of flukes for several days to weeks, such as for or RNA interference-mediated knockdown of gene transcripts, which again needs a reliable quantification of expression. For the relative quantification of gene expression by quantitative real-time PCR (qRT-PCR), the accuracy is largely dependent on the stable expression of the reference genes used for normalisation. However, classical housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (gapdh) and ribosomal RNA (rRNA) are often used based on tradition rather than being experimentally validated as stably expressed genes for the species or parasite stage of interest.

In this study, we defined a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We compared the expression stabilities of eight candidate reference genes, pre-selected from transcriptome data, by the algorithms NormFinder, geNorm, BestKeeper, and comparative DCT 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 of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes may be suited to replace the housekeeping gene gapdh which has been used as standard in most Fasciola studies to date, but in fact was differentially expressed in our analysis.

As a first application of the newly defined reference genes, we determined the expression levels of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) which are discussed as potential druggable targets in other parasitic flatworms. Distinct expression patterns throughout development were found pointing to interesting biological functions, such as a predominant expression of the polo-like 1 kinase in adult worms. Our results may motivate using this set of validated reference genes for future studies with Fasciola dealing with drug targets or parasite development.