快速荧光<em>原位</em>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
Summary
We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.
Abstract
原位杂交（FISH）用免疫荧光（免疫FISH）结合的RNA的荧光创建可以在单细胞水平被用于检测RNA的定位与同步洞察的空间动态成 蛋白质，表观遗传修饰和其他细节的定位技术这可以通过免疫荧光被突出显示。 X染色体失活是一个范例长非编码RNA（lncRNA）介导的基因沉默。的X不活动的特定转录物（Xist的）lncRNA积累（称为Xist的云）上的两个X染色体在哺乳动物雌性之一是启动的X染色体失活的一个关键步骤。 Xist的核糖核酸直接或间接地与各种染色质修饰酶相互作用并引入不同的后生景观的失活的X染色体（十一）。在西安一个已知的后生特点是组蛋白H3三甲基赖氨酸27（H3K27me3）修改。在这里，我们描述了一个简单而快速的免疫FISH协议利用RNA FISH配上H3K27me3的免疫多个寡核苷酸探针检查Xist的RNA的定位及相关表观遗传修饰检测Xist的RNA。使用寡核苷酸探针的结果在较短的培养时间和更灵敏的检测Xist的核糖核酸的相比在体外转录的RNA探针（核糖探针）。这个协议提供了一个有力的工具用于理解lncRNAs的动力学和其相关联的表观遗传修饰，染色质结构，核组织和转录调控。
Introduction
哺乳动物的X染色体失活（XCI）是一种策略，以补偿在XX和XY，其中两个X染色体在女性中的一个是转录失活的1之间的X连锁的基因剂量的不平衡。 X染色体失活的长非编码RNA（lncRNA）的研究有很大的模型系统。的X染色体失活是由多个lncRNAs调节，并且已被广泛研究，在过去的几十年中，以揭开lncRNAs，转录，染色质结构和核组织2，3之间的串扰的机制。
位于X染色体的X染色体失活中心（XIC）是一个复杂的遗传基因座由多个基因产生的非编码RNA 4。的X不活动的特定转录物（Xist的）lncRNA在真哺乳亚纲哺乳动物是一个这样的lncRNA起着在X-染色体失活-5,6-了至关重要的作用。 Xist的成绩单环绕夫的位置TURE习近平启动X染色体失活，而当使用RNA FISH可视化显示为云;此形成被称为“Xist的云”7。由于Xist的RNA与各种染色质修饰酶相互作用，共同定位的Xist的云与沉默染色质和镇压转录不同的表观遗传修饰的是在X染色体失活8观察。例如，Xist的RNA相互作用的Polycomb镇压复杂2（PRC2）负责H3K27me3和诱导镇压染色质状态9。在Xist的云兮其共定位与密集的H3K27me3修饰的发生表示曦10,11的兼异的风景线。
细胞遗传学技术，如DNA / RNA FISH用放射性标记探针12最近的和先进的技术，具有增强的灵敏度，已经走过了漫长的道路，从传统的方法荧光成像使用多个寡核苷酸探针13,14。的DNA / RNA的FISH加上免疫已被常规地用作细胞学工具理解时空核组织的RNA定位，染色质结构和修改。最标准的探针制备用于RNA FISH涉及使用质粒或细菌人工染色体（BAC）克隆并随后将其标识或者与切口平移或随机引物15的。然而，近70％的基因在小鼠和基因在人类中的40％的显示的正义和反义转录物16的重叠，因此需要一个链特异的FISH方法以区分义和反义转录物， 在体外转录的RNA探针（核糖探针）通常用于链特异的RNA原位杂交17,18;然而，这涉及到一种制备质粒克隆或PCR产物用T7，SP6中，或T3启动子和合成的核糖核酸探针。此外，核糖探针从GENOM派生IC DNA或cDNA常含有非特定区域和重复的元素，这导致高的背景噪声。另一个问题是，核糖核酸探针，它们是几百个核苷酸的长度，并包含多个荧光团，不能有效地渗透到细胞核中。为了规避这一点，标有在末端的单个荧光多个较短的寡核苷酸探针已经开发出具有良好的灵敏度，均匀的信号强度，并且易于纯化和处理14。此外，DNA寡核苷酸通常比核糖核酸更稳定。我们应用使用Xist的RNA FISH 19寡核苷酸与免疫，了解西安的X染色体失活的过程中诱导Xist的lncRNA的后生动态类似的策略。这个协议描述的创建的寡核苷酸探针和细胞的适当的准备，以及利用免疫荧光和RNA原位杂交的。 Xist的RNA FISH使用多oligonucleotides是在长期成本效益的方法，如果Xist的RNA FISH在自己的实验室常规进行。该技术可用于识别lncRNAs在细胞中同时映射其共定位与表观遗传修饰或因素。该协议的一个主要优点是可以轻松地修改它以满足自己的研究兴趣的能力。
Protocol
Representative Results
Discussion
在本文中，我们提出了一个快速的免疫FISH协议，只需不到5小时即可完成幻灯片的准备，Xist的RNA FISH，和H3K27me3免疫。在与Xist的RNA检测，这通常需要过夜孵育的RNA原位杂交的一般性免疫FISH方法相比，该协议不仅显著降低所花费的时间，而且，提高了使用的寡核苷酸探针的免疫-FISH的灵敏度。
因为Xist的期间的X染色体失活高度转录和其转录被发现强烈聚积在希在顺式 ，很…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children’s Hospital Medical Center to Y.O.
Materials
| oligonucleotides with 5’-amine modification | IDT | |
| Alexa Fluor 488 Reactive Dye | Life Technologies | A32750 |
| MicroSpin G-25 columns | GE Healthcare | 27-5325-01 |
| Cytospin 2 | Shandon | 59900102 |
| Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer) | Roche | 10715859103 |
| Histone H3K27me3 antibody | Active Motif | 61017 |
| Accutase | Innovative Cell Technologies | AT104 |
| Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed | Life Technologies | A21424 |
| ProLong Gold antifade reagent | Life Technologies | P36931 |
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