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Defining an optimal control for RNAi experiments with adult Schistosoma mansoni
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 agreem...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10276032/ https://www.ncbi.nlm.nih.gov/pubmed/37328492 http://dx.doi.org/10.1038/s41598-023-36826-6 |
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author | Moescheid, Max F. Puckelwaldt, Oliver Beutler, Mandy Haeberlein, Simone Grevelding, Christoph G. |
author_facet | Moescheid, Max F. Puckelwaldt, Oliver Beutler, Mandy Haeberlein, Simone Grevelding, Christoph G. |
author_sort | Moescheid, Max F. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-10276032 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-102760322023-06-18 Defining an optimal control for RNAi experiments with adult Schistosoma mansoni Moescheid, Max F. Puckelwaldt, Oliver Beutler, Mandy Haeberlein, Simone Grevelding, Christoph G. Sci Rep Article 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. Nature Publishing Group UK 2023-06-16 /pmc/articles/PMC10276032/ /pubmed/37328492 http://dx.doi.org/10.1038/s41598-023-36826-6 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Moescheid, Max F. Puckelwaldt, Oliver Beutler, Mandy Haeberlein, Simone Grevelding, Christoph G. Defining an optimal control for RNAi experiments with adult Schistosoma mansoni |
title | Defining an optimal control for RNAi experiments with adult Schistosoma mansoni |
title_full | Defining an optimal control for RNAi experiments with adult Schistosoma mansoni |
title_fullStr | Defining an optimal control for RNAi experiments with adult Schistosoma mansoni |
title_full_unstemmed | Defining an optimal control for RNAi experiments with adult Schistosoma mansoni |
title_short | Defining an optimal control for RNAi experiments with adult Schistosoma mansoni |
title_sort | defining an optimal control for rnai experiments with adult schistosoma mansoni |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10276032/ https://www.ncbi.nlm.nih.gov/pubmed/37328492 http://dx.doi.org/10.1038/s41598-023-36826-6 |
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