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.

Rhodopsin orphan GPCR20 interacts with neuropeptides and directs growth, sexual differentiation, and egg production in female Schistosoma mansoni

“…Since a high amount of dsRNA (30 µg/mL for Smgpcr20 and 15 µg/mL for Smnpp26 and Smnpp40 , respectively) was used for the triple KD approach, we performed another control experiment to exclude that treatment with this high amount of dsRNA may negatively influence our results. To this end, we made use of dsRNA of the ampR gene of Escherichia coli , which in a recent study was shown to be suitable as “irrelevant” control dsRNA in RNAi experiments with S. mansoni ( 46 ). Since in the mentioned study, 30 µg/mL dsRNA had been used, we repeated the experiment here with 60 µg/mL dsRNA.…”

ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT ASCHISTOSOMA MANSONIGENOME SAFE HARBOR

“…For this, the external standard curve design was employed to estimate integrated-EGFP in gDNA of the schistosome eggs (n = 3) as described (26). The standard curve is based on linear regression (y = 3.3857x+35.093, R 2 = 0.9979) established by a logarithm (20)-transformed initial DNA input, the pJCR53.2-SmUbi-EGFP-SmUbi plasmid was used as the dependent and the Ct value from the qPCRs as the independent variation (not shown). We estimated the EGFP transgene copy number in GSH1 by converting the observed Ct values to the logarithmic copy numbers using the equations obtained from the standard curve.…”

“…Until now, RNA interference (RNAi) has been proven as the most suitable method for functional gene characterization. However, RNAi efficiency varies, and it can lead to ectopic effects (17)(18)(19)(20). To study GOI, knock-out (KO) models are common for various model organisms, but not yet established for trematodes or other helminths.…”

An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum

“…Firstly, we employed gfp dsRNA as the control. Previous research on S. mansoni has indicated that gfp can cause changes in the expression of related genes [ 51 ]. Therefore, it is important to carefully choose appropriate controls that have been used in S. japonicum .…”