Yue Minghui, Charles Richard John Lalith, Yamada Norishige, Ogawa Akiyo, Ogawa Yuya
Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center; Department of Pediatrics, University of Cincinnati College of Medicine.
Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center.
J Vis Exp. 2014 Nov 26(93):e52053. doi: 10.3791/52053.
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.
将RNA荧光原位杂交（FISH）与免疫荧光相结合（免疫FISH），创造了一种可在单细胞水平上应用的技术，用于检测RNA定位的空间动态，同时深入了解蛋白质的定位、表观遗传修饰以及其他可通过免疫荧光突出显示的细节。X染色体失活是长链非编码RNA（lncRNA）介导的基因沉默的一个范例。在哺乳动物雌性个体的两条X染色体之一上，X失活特异性转录本（Xist）lncRNA的积累（称为Xist云）是启动X染色体失活的关键步骤。Xist RNA直接或间接与各种染色质修饰酶相互作用，并为失活的X染色体（Xi）引入独特的表观遗传景观。Xi的一个已知表观遗传特征是组蛋白H3三甲基赖氨酸27（H3K27me3）修饰。在这里，我们描述了一种简单快速的免疫FISH方案，用于使用RNA FISH和多个寡核苷酸探针检测Xist RNA，并结合H3K27me3的免疫荧光来检查Xist RNA的定位和相关的表观遗传修饰。与体外转录的RNA探针（核糖探针）相比，使用寡核苷酸探针可缩短孵育时间并更灵敏地检测Xist RNA。该方案为理解lncRNAs的动态及其相关的表观遗传修饰、染色质结构、核组织和转录调控提供了一个强大的工具。
