Detection of Long Non-Coding RNA Localization by RNA Fluorescence In Situ Hybridization (FISH).

Long non-coding RNAs (lncRNAs) regulate a wide range of important biological processes through their RNA molecules, yet the majority remain poorly understood at the functional level. Because subcellular localization plays a critical role in lncRNA function, determining spatial distribution is essential for understanding their mechanisms of action. To this end, RNA fluorescence in situ hybridization (RNA FISH) enables direct visualization of RNA molecules with single-cell spatial resolution. Here, we present a robust RNA FISH protocol for detecting the lncRNA Xist, a chromatin-associated master regulator of X chromosome inactivation (XCI) that serves as a paradigm for lncRNA biology. Using this protocol, we observe the characteristic Xist clouds on the inactive X chromosomes in mouse embryonic fibroblasts (MEFs), demonstrating the effectiveness of this method for investigating lncRNA localization and chromatin-associated function.