Defining an optimal control for RNAi experiments with adult Schistosoma mansoni

Abstract

In parasites such as Schistosoma mansoni, gene knockdown by RNA interference (RNAi) has become an indispensable tool for functional gene characterization. To distinguish target-specific RNAi effects versus off-target effects, controls are essential. To date, however, there is still no general agreement about suitable RNAi controls, which limits the comparability between studies. To address this point, we investigated three selected dsRNAs for their suitability as RNAi controls in experiments with adult S. mansoni in vitro. Two dsRNAs were of bacterial origin, the neomycin resistance gene (neoR) and the ampicillin resistance gene (ampR). The third one, the green fluorescent protein gene (gfp), originated from jellyfish. Following dsRNA application, we analyzed physiological parameters like pairing stability, motility, and egg production as well as morphological integrity. Furthermore, using RT-qPCR we evaluated the potential of the used dsRNAs to influence transcript patterns of off-target genes, which had been predicted by si-Fi (siRNA-Finder). At the physiological and morphological levels, we observed no obvious changes in the dsRNA treatment groups compared to an untreated control. However, we detected remarkable differences at the transcript level of gene expression. Amongst the three tested candidates, we suggest dsRNA of the E. coli ampR gene as the most suitable RNAi control.