Quick Fluorescent <em>In Situ</em> Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Minghui Yue; John Lalith Charles Richard; Norishige Yamada; Akiyo Ogawa; Yuya Ogawa

doi:10.3791/52053

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Biology

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Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

快速荧光原位对于Xist的RNA杂交组合议定书组蛋白修饰免疫荧光X染色体失活

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12:42 min

November 26, 2014

DOI: 10.3791/52053-v

Minghui Yue*^1,2, John Lalith Charles Richard*^1,2, Norishige Yamada^1,2, Akiyo Ogawa¹, Yuya Ogawa^1,2

¹Division of Reproductive Sciences,Cincinnati Children's Hospital Medical Center, ²Department of Pediatrics,University of Cincinnati College of Medicine

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我们开发了一种易于定制的链特异性荧光原位杂交（FISH）方案与免疫荧光相结合。这允许详细检查 RNA 动力学，同时在单细胞水平上了解染色质结构、核组织和转录调控。

该程序的总体目标是通过使用快速荧光原位杂交结合免疫荧光来研究 X 染色体失活过程中长链非编码存在 RNA 及其相关表观遗传修饰的动力学。该方案需要制备带有分化胚胎体细胞的载玻片的胞嘧啶，这些载玻片被渗透然后固定用于染色。接下来，由组蛋白 H 三甲基赖氨酸 27 抗体定位的荧光标记物在失活的 X 染色体上积累。

最后一步使用寡核苷酸探针，该探针包含五个主要氨基修饰，并用 AM 平均反应性荧光团标记以执行存在。RNA 鱼。最终，荧光显微镜用于显示 E Es 细胞分化过程中存在的 RNA 云和组蛋白修饰的定位。

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