Protocol to assess RNA-RNA interactions in situ using an RNA-proximity ligation assay

Here, we describe a protocol to assess RNA-RNA interactions in situ using an adapted proximity ligation assay (PLA). We detail steps to perform RNA-probe hybridization, in situ rolling circle amplification, and immunofluorescence confocal microscopy. With these tools, it is possible to detect and characterize the intracellular localization of interacting RNA pairs using small cell numbers. This protocol provides a targeted approach to understanding RNA-RNA interactions in intact cells that can complement other established deep-sequencing-based approaches. For complete details on the use and execution of this protocol, please refer to Basavappa et al. (2022).¹.