Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay

RNA transcribed at DNA double-strand breaks (DSBs) contributes to accurate DNA repair. Here, using the repair factors 53BP1 and TIRR as examples, we combine the fluorescence in situ hybridization (FISH) and proximity ligation assay (PLA) techniques to determine protein proximity to DSB-transcribed R...

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Autores principales: Alagia, Adele, Ketley, Ruth F., Gullerova, Monika
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9932567/
https://www.ncbi.nlm.nih.gov/pubmed/36825808
http://dx.doi.org/10.1016/j.xpro.2023.102096
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author Alagia, Adele
Ketley, Ruth F.
Gullerova, Monika
author_facet Alagia, Adele
Ketley, Ruth F.
Gullerova, Monika
author_sort Alagia, Adele
collection PubMed
description RNA transcribed at DNA double-strand breaks (DSBs) contributes to accurate DNA repair. Here, using the repair factors 53BP1 and TIRR as examples, we combine the fluorescence in situ hybridization (FISH) and proximity ligation assay (PLA) techniques to determine protein proximity to DSB-transcribed RNA. In this FISH-PLA protocol, we detail steps for designing DNA probes and image analysis using CellProfiler™ software. This approach has many potential applications for the study of the RNA-binding proteins and nascent RNA interactions. For complete details on the use and execution of this protocol, please refer to Ketley et al. (2022).(1)
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spelling pubmed-99325672023-02-17 Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay Alagia, Adele Ketley, Ruth F. Gullerova, Monika STAR Protoc Protocol RNA transcribed at DNA double-strand breaks (DSBs) contributes to accurate DNA repair. Here, using the repair factors 53BP1 and TIRR as examples, we combine the fluorescence in situ hybridization (FISH) and proximity ligation assay (PLA) techniques to determine protein proximity to DSB-transcribed RNA. In this FISH-PLA protocol, we detail steps for designing DNA probes and image analysis using CellProfiler™ software. This approach has many potential applications for the study of the RNA-binding proteins and nascent RNA interactions. For complete details on the use and execution of this protocol, please refer to Ketley et al. (2022).(1) Elsevier 2023-02-03 /pmc/articles/PMC9932567/ /pubmed/36825808 http://dx.doi.org/10.1016/j.xpro.2023.102096 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Alagia, Adele
Ketley, Ruth F.
Gullerova, Monika
Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay
title Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay
title_full Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay
title_fullStr Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay
title_full_unstemmed Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay
title_short Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay
title_sort detecting rna-protein proximity at dna double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9932567/
https://www.ncbi.nlm.nih.gov/pubmed/36825808
http://dx.doi.org/10.1016/j.xpro.2023.102096
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