Visualizing Long Noncoding RNAs on Chromatin

Abstract

Fluorescence in situ hybridization (FISH) enables the detection of specific nucleic acid sequences within single cells. For example, RNA FISH provides information on both the expression level and localization of RNA transcripts and, when combined with detection of associated proteins and chromatin modifications, can lend essential insights into long noncoding RNA (lncRNA) function. Epigenetic effects have been postulated for many lncRNAs, but shown for only a few. Advances in in situ techniques and microscopy, however, now allow for visualization of lncRNAs that are expressed at very low levels or are not very stable. FISH-based detections of RNA and DNA coupled with immunological staining of proteins/histone modifications offer the possibility to connect lncRNAs to epigenetic effects. Here, we describe an integrated set of protocols to detect, individually or in combination, specific RNAs, DNAs, proteins, and histone modifications in single cells at a high level of sensitivity using conventional fluorescence microscopy.

Keywords: Chromatin; DNA FISH; Epigenetic; Fluorescence in situ hybridization; Histone modifications; Immunofluorescence; Long noncoding RNAs; RNA FISH.

Fig. 1

Immunofluorescence followed by strand-specific RNA FISH in female mouse trophoblast stem cells. Histone H3 lysine 27 trimethylation (H3-K27me3; in green) is enriched on the inactive X-chromosome that is marked by Xist lncRNA accumulation (purple). The active X-chromosome is marked by nascent expression of the Xist antisense Tsix lncRNA (red pinpoints). Nuclei are stained blue with DAPI

Fig. 2

RNA followed by DNA FISH in female mouse epiblast stem cells. Strand-specific RNA FISH detection of Xist lncRNA (white) and the Xist antisense Tsix lncRNA (green) followed by DNA FISH detection of the Xist/Tsix locus. Nuclei are stained blue with DAPI