Visualizing Long Noncoding RNAs on Chromatin
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.
Key wordsImmunofluorescence Fluorescence in situ hybridization RNA FISH DNA FISH Long noncoding RNAs Epigenetic Chromatin Histone modifications
This work was funded by an NIH National Research Service Award #5-T32-GM07544 from the National Institute of General Medicine Sciences to E.M.; an NIH Director’s New Innovator Award (DP2-OD-008646-01) to S.K.; a March of Dimes Basil O’Connor Starter Scholar Research Award (5-FY12-119); and the University of Michigan Endowment for Basic Sciences.