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

Adele Alagia; Ruth F Ketley; Monika Gullerova

doi:10.1016/j.xpro.2023.102096

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

STAR Protoc. 2023 Mar 17;4(1):102096. doi: 10.1016/j.xpro.2023.102096. Epub 2023 Feb 3.

Authors

Adele Alagia¹, Ruth F Ketley², Monika Gullerova³

Affiliations

¹ Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address: adele.alagia@path.ox.ac.uk.
² Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
³ Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address: monika.gullerova@path.ox.ac.uk.

Abstract

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.

Keywords: Cell Biology; In Situ Hybridization; Microscopy; Molecular Biology; Molecular/Chemical Probes.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

DNA / metabolism
DNA Breaks, Double-Stranded*
In Situ Hybridization, Fluorescence
RNA* / genetics
RNA-Binding Proteins / genetics

Substances

RNA
RNA-Binding Proteins
DNA

Grants and funding

24866/CRUK_/Cancer Research UK/United Kingdom