title: Quick fluorescent in situ hybridization protocol for Xist RNA combined with immunofluorescence of histone modification in X-chromosome inactivation.
abstract: Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.
authors.affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center; Department of Pediatrics, University of Cincinnati College of Medicine.
authors.forename: Minghui
authors.initials: M
authors.lastname: Yue
authors.affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center; Department of Pediatrics, University of Cincinnati College of Medicine.
authors.forename: John Lalith
authors.initials: JL
authors.lastname: Charles Richard
authors.affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center; Department of Pediatrics, University of Cincinnati College of Medicine.
authors.forename: Norishige
authors.initials: N
authors.lastname: Yamada
authors.affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center.
authors.forename: Akiyo
authors.initials: A
authors.lastname: Ogawa
authors.affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center; Department of Pediatrics, University of Cincinnati College of Medicine; Yuya.Ogawa@cchmc.org.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Ogawa
mesh_terms: D000818:Animals; D053595:Embryonic Stem Cells; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D006657:Histones / Q000737:chemistry / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000235:genetics*; D014960:X Chromosome; D049951:X Chromosome Inactivation
chemical_list: D006657:Histones; D062085:RNA, Long Noncoding; C077885:XIST non-coding RNA