The stability and sequence cleavage preference of dsRNA are key factors differentiating RNAi efficiency between migratory locust and Asian corn borer

Fan, Yunhe; Song, Huifang; Abbas, Mureed; Wang, Yanli; Liu, Xiaojian; Li, Tao; Ma, Enbo; Zhu, Kun Yan; Zhang, Jianzhen

doi:10.1016/j.ibmb.2022.103738

Cited by 10 publications

(5 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The influence of GC content or thermodynamic stability is a described phenomenon 29 . Other known aspects are the influences of sequence cleavage preference in dsRNA processing by dicer, which was described in Paramecium, moths, or grasshoppers 69 , 70 . As to date, there is no detailed characterization of the RNAi machinery in schistosomes, thus further work is needed to determine the influence of sequence composition of dsRNAs on RNAi efficacy in this parasite.…”

Section: Discussionmentioning

confidence: 99%

Defining an optimal control for RNAi experiments with adult Schistosoma mansoni

Moescheid,

Puckelwaldt,

Beutler

et al. 2023

Sci Rep

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Defining an optimal control for RNAi experiments with adult Schistosoma mansoni

Moescheid,

Puckelwaldt,

Beutler

et al. 2023

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Since injection of ds EGFP could induce a strong response of OfDicer2 and OfAgo2 , OfEF1 α and OfCTP8 were selected as two target genes to determine whether the silencing efficiency against the two target genes could be improved by pre-injection of ds EGFP . Previous studies have shown that OfEF1 α can be significantly silenced by tissue culture RNAi in O. furnacalis [ 40 ], and OfCTP8 can be significantly knocked down by injection of ds OfCTP8 in O. furnacalis [ 13 ]. As expected, OfEF1 α could not be significantly silenced in any tissues (hemolymph, integument, and midgut) when O. furnacalis larvae were pre-injected with ddH 2 O.…”

Section: Discussionmentioning

confidence: 99%

Increased RNAi Efficiency by dsEGFP-Induced Up-Regulation of Two Core RNAi Pathway Genes (OfDicer2 and OfAgo2) in the Asian Corn Borer (Ostrinia furnacalis)

Fan

Abbas

Liu

et al. 2022

Insects

Self Cite

View full text Add to dashboard Cite

RNA interference (RNAi) is a sequence-specific gene silencing mechanism that holds great promise for effective management of agricultural pests. Previous studies have shown that the efficacy of RNAi varies among different insect species, which limits its wide spread application in the field of crop protection. In this study, we identified and characterized six core RNAi pathway genes including OfDicer1, OfDicer2, OfR2D2, OfAgo1, OfAgo2, and OfAgo3 from the transcriptomic database of the Asian corn borer (Ostrinia furnacalis). Domain analysis showed that the six deduced proteins contained the necessary functional domains. Insect developmental stage- and tissue-specific expression analysis showed that five genes were expressed in all the stages and tissues examined except OfAgo3, which showed low expression in larvae, and high expression in pupae and adults and in the midgut. RT-qPCR was performed to examine the response of these six genes to exogenous double-stranded RNA (dsRNA). Interestingly, the transcript levels of OfDicer2 and OfAgo2 were significantly enhanced after the injection of dsEGFP at different time points and tissues investigated. Consequently, the RNAi efficiency in targeting the insect endogenous genes can be greatly enhanced in the hemolymph or midgut. Taken together, our investigations suggest that RNAi efficiency can be enhanced by pre-injection of dsRNA to induce the RNAi core machinery genes, which could be a useful strategy to improving RNAi efficiency for studying gene functions under laboratory conditions.

show abstract

“…For example, in C. elegans and many insects, short dsRNAs (<40 bps) are not effectively taken up by cells (e.g., Clemens et al, 2000;Miller et al, 2012), so designing long dsRNA constructs (e.g., 200-500 bp) is important for ensuring a strong and stable knockdown. In addition, in some species dsRNA processing (i.e., cleavage into siRNAs, see Section 3.2) is affected by the dsRNA sequence itself (Fan et al, 2022). It is therefore often useful to design and test a variety of constructs for a single target gene (e.g., He et al, 2020).…”

Section: Optimizing Your Rnai Protocolmentioning

confidence: 99%