Nuclear-enriched long non-coding RNAs (lncRNAs) can regulate gene expression by interacting with specific RNA-binding proteins (RBPs). Here, we present a protocol to directly image these lncRNAs at the single-molecule level together with their protein interactors in human cardiomyocytes. We describe steps to perform RNA-fluorescence in situ hybridisation (FISH) of a nuclear intron-retaining lncRNA, followed by sequential protein immunofluorescence (IF), enabling simultaneous visualisation of specific RNA and protein targets. We also detail procedures for probe design, cell seeding, and fixation.
Buonaiuto et al. (Thu,) studied this question.