Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells
Summary: Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722004762 |
| _version_ | 1818179100054913024 |
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| author | Yajie Gao Yuanxiang Zhu Qinmiao Sun Dahua Chen |
| author_facet | Yajie Gao Yuanxiang Zhu Qinmiao Sun Dahua Chen |
| author_sort | Yajie Gao |
| collection | DOAJ |
| description | Summary: Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5′- and 3′-cleavage products simultaneously.For complete details on the use and execution of this protocol, please refer to Gao et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-12-11T20:58:30Z |
| format | Article |
| id | doaj.art-710b52942f9c44d592152fe9a1a9daa6 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-11T20:58:30Z |
| publishDate | 2022-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-710b52942f9c44d592152fe9a1a9daa62022-12-22T00:51:03ZengElsevierSTAR Protocols2666-16672022-09-0133101596Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cellsYajie Gao0Yuanxiang Zhu1Qinmiao Sun2Dahua Chen3Institute of Biomedical Research, Yunnan University, Kunming 650500, China; Corresponding authorInstitute of Biomedical Research, Yunnan University, Kunming 650500, ChinaState Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute of Stem Cells and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Corresponding authorInstitute of Biomedical Research, Yunnan University, Kunming 650500, China; State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; CorrespondenceSummary: Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5′- and 3′-cleavage products simultaneously.For complete details on the use and execution of this protocol, please refer to Gao et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722004762Cell-based AssaysMolecular BiologyGene ExpressionMolecular/Chemical ProbesProtein Biochemistry |
| spellingShingle | Yajie Gao Yuanxiang Zhu Qinmiao Sun Dahua Chen Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells STAR Protocols Cell-based Assays Molecular Biology Gene Expression Molecular/Chemical Probes Protein Biochemistry |
| title | Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells |
| title_full | Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells |
| title_fullStr | Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells |
| title_full_unstemmed | Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells |
| title_short | Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells |
| title_sort | optimized protocols for rna induced silencing complex assembly and cleavage in cultured drosophila cells |
| topic | Cell-based Assays Molecular Biology Gene Expression Molecular/Chemical Probes Protein Biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722004762 |
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