这项工作是支持的，CA131967美国国家癌症研究所的资助。

1。 BrdU掺入（末端标记） <ol><li>至约70％汇合在一个150毫米的组织培养皿中的24小时之前加入的BrdU板细胞。 </li><li>用新鲜的完整的媒体，含有20微克/毫升的BrdU（Sigma公司）在适当的时间点，在收获之前更换介质。用BrdU与细胞类型和种类会有所不同，典型的G2期的持续时间在2和5小时（ 图1）之间的介质中培养时细胞的长度。 </li></ol> 2。单层细胞培养染色体的收获<ol><li>删除的文化传媒板块，并收集10毫升，15毫升的离心管中。丢弃剩余的介质。 </li><li>冲洗细胞一次用10ml正矢[或HBSS中（Sigma公司）]。 </li><li>加入5毫升正矢（或HBSS中）中的0.25％胰蛋白酶的板。在室温下孵育，直到细胞脱离板。 </li><li>加入胰蛋白酶处理的细胞悬浮液从步骤2.1的介质中含有10毫升的管。&lt;/ P&gt; <li>离心400 xg离心10分钟以沉淀细胞。吸干上清液，离开0.5毫升介质中加细胞沉淀。 </li><li>用巴斯德吸管移液彻底重悬细胞。 </li><li>加入3滴低渗溶液（0.075 M氯化钾（Sigma公司）温热至37℃下 ），用巴斯德移液管中的细胞悬浮液混合。加入0.5毫升低渗溶液混合，再次用巴斯德吸管。带来的低渗溶液至5毫升的量，并再次混合。在37℃下孵育20-45分钟，这取决于细胞的类型。 </li><li> 400×g离心10分钟，离心。吸干上清液，离开0.5毫升介质中加细胞沉淀。 </li><li>重悬细胞沉淀轻轻轻拂管。在这一点上，渗透细胞肿胀是脆弱的，所以应注意不破坏细胞膜。 </li><li>加3滴混合，轻轻移液用巴斯德pipett卡诺固定液（1:3的冰醋酸酸:甲醇）;E。加入0.5毫升的固定剂和组合，用巴氏吸管轻轻吹打。带上固定液至5毫升的体积，并再次混合，轻轻移液用巴斯德移液管。固定的细胞可以被存储在黑暗中在-20℃下的数个月。 </li><li>离心并吸出，在步骤2.8。 </li><li>细胞沉淀重悬在新鲜固定液;估计的体积的颗粒和附加组件〜10倍的体积的卡诺固定液。 </li><li>添加的细胞悬浮液，逐滴湿，将冰冷的显微镜载玻片上，将载玻片固定在一个〜45°角，将细胞悬浮液，并允许流过的表面的滑动。莱平放在纸巾滑动和洪水的幻灯片，固色剂，在空气中晾干。检查存在的有丝分裂利差用倒置显微镜幻灯片。 </li></ol> 3。核糖核酸酶和乙醇脱水<ol><li>将200μl的10微克/毫升核糖核酸酶A（Sigma）的2倍SSC到每张幻灯片孵育在37 和德克，C 1小时。 </li><li>洗涤滑动3次，用2×SSC，每次3分钟，在室温下的pH值为7.0。 </li><li>通过乙醇（Sigma）的系列（70％，90％和100％），在室温下，每次3分钟脱水幻灯片。 </li><li>空气干燥，在室温下滑动。 </li></ol> 4。 原位杂交探针鸡尾酒BAC加特定染色体着丝粒的计数探针（CEP）或整条染色体涂料的制备<ol><li>对于BAC / CEP同时杂交预杂交，准备两个单独的探头鸡尾酒。我们已经发现，预杂交BAC和CEP探针分别根据不同的严谨的查询结果在以下的背景和更大的信号强度。我们还发现，也可以用来代替BAC克隆个别F粘粒。下面的鸡尾酒配方代表一个单一的幻灯片的体积。如果处理多张幻灯片使用相同的探头简单地增加销量appropr<html> iately。 * BAC DNA探针鸡尾酒: 11.5微升杂交缓冲液（50％甲酰胺（Sigma公司）/ 2个SSC /硫酸葡聚糖（Sigma公司））2μL蒸馏水2 O 2.5微升BAC/Cot1 DNA在DH 2 O（见下面的标签协议）总体积15μL * CEP探头鸡尾酒: 7微升CEP杂交缓冲液（65％Formamide/2x SSC /葡聚糖硫酸酯）2微升DH 2 O 1的微升CEP/Cot1 DNA（与频谱Orange/Cy3/Texas红prelabeled购买）10μl总体积 *在变性和预杂交（5.1）混合BAC探针与CEP探头在3:2的比例25μL/幻灯片和混合探针鸡尾酒。 或</li><li>等分试样20微升/滑动的的全染色体涂料探针（Cy3/Texas红色）成管。继续变性和预杂交步骤。 </li></ol>"&gt; 5。幻灯片和探头变性和原位杂交<ol><li>变性探针鸡尾酒（s）在75℃下 10分钟。预杂交在37℃下30分钟，以允许Cot1的DNA重复序列的杂交，以防止随后的杂交到中期染色体。 </li><li>变性幻灯片科普林氏罐子在70％Formamide/2x SSC，pH值7.0在72°C，持续3分钟。 </li><li>立即脱水幻灯片通过科普林氏罐子中的乙醇系列（70％，90％和100％），在4℃下，每次3分钟。 </li><li>允许的幻灯片空气干燥，在室温下。 </li><li>预暖的幻灯片至45℃，幻灯片上的探针的预杂交（步骤5.1）的最后10分钟的温暖。如果需要预杂交探针鸡尾酒混合，并加入25微升到每张幻灯片，轻轻将每张幻灯片上，并沿边缘用橡皮泥密封盖玻片。在37℃下孵育过夜，在潮湿的腔室，以防止蒸发。 </li></ol><ol><li>洗净滑动科普林氏罐3次，每次3分钟，在38-40℃下在pH值为7.0×SSC，50％formamide/2x。的洗涤液的温度，可能会发生变化的探针之间。如果有高背景杂交可以增加的洗涤液的温度。反之，如果信号微弱，没有任何背景，那么你可以减少这些清洗的温度。 </li><li>洗幻灯片1 PN缓冲的时间，在室温下 3分钟，然后继续的BrdU检测步骤。 </li></ol> 7。的BrdU检测<ol><li>封锁PNM缓冲液在室温下在黑暗中10分钟（200微升/幻灯片）的幻灯片。 </li><li>排水幻灯片，打开边缘，在纸巾上。加入100微升抗BrdU-FITC（50μg/ ml的罗氏或Millipore）在PNM缓冲液30分钟，在37℃下。 </li><li>洗净幻灯片PN缓冲液中，在室温下的3次，每次3分钟。 </li><li>排水过剩的PN缓冲滑动打开边缘纸巾上的时间。加入20μlDAPI /的抗褪色安装的解决方案（Invitrogen公司）。 量的幻灯片用纸巾摁倒在盖玻片给逼出来的气泡和多余的安装解决方案。进行图像分析。 </li></ol> 8。捕捉图像和量化BrdU掺入<ol><li>与奥林巴斯BX61荧光显微镜和CCD相机奥林巴斯100倍的目标，自动过滤器轮和"Cytovision软件（应用影像）拍摄的图像。尿嘧啶被捕捉使用的FITC过滤器;染色体油漆（德克萨斯红，Metasystems或Cytocell）的，BAC的（Cy3标记）和CEP（Vysis频谱橙色）探头被捕获使用一个得克萨斯州红或Cy3的过滤器; DAPI被抓获一个DAPI过滤器（见图2和图3）。 </li><li>个人感兴趣的染色体识别中的BAC / CEP或染色体特异性漆探针（ 图2B和图3B）。利用Cytovision软件，感兴趣的每个染色体是"切出"作为一个整体从中期扩展，一条线绘制通过沿其长度从短臂的染色体长臂（ 图2C和3C）。 Cytovision软件用于沿每个染色体的长度，以量化的像素强度的档案。 Cytovision软件计算的象素所占用的面积，和量化的BrdU或DAPI每个隔离染色体上的信号表示的像素的强度。的那些像素所占据的面积，以获得总的像素数（ 表1和图4），然后乘以每个染色体所表示的平均像素强度。此外，该协议允许最新的复制的染色体区域的可视化。因此，条带状图案的BrdU掺入允许用于检测染色体积极复制区域，并且在复制中的定时的差异之间彩色石印青梅对被视为在此带型（ 图2和图3）的差异。最后，在结合模式的这些差异可以比已知的每个染色体6,7，它允许对同源染色体或染色体臂4之间的定时差的估计值的复制的复制时间地图。例如，两个染色体6的在图3A中显示的差&gt; 2小时的带型时相比，第6号染色体4正常的复制定时。 此外，此处所述的相似的方法已被成功地用于研究的异步复制定时之间的无效和有效的X染色体8,9。确定了使用X染色体涂料的活性和失活的X染色体。画像购入使用QUIPS mFISH软件（Vysis），和X染色体所占据的像素的数目和由荧光拉占用的像素的数目与等量的BrdU，然后计算使用NIH图像（ <a href="http://rsb.info.nih.gov/nihimage" target="_blank">http://rsb.info.nih.gov/nih-image/index.html</a> ）的。 </li></ol> 9。尼克翻译BAC DNA荧光标记（Vysis） <ol><li>下面的探针混合物冷却微型离心管: 70微升（4微克DNA）在卫生署2 O 10微升0.2毫米的dUTP，橙色或绿色 20微升0.1毫米dTTP的 20微升10倍尼克翻译缓冲液 40微升NT的dNTP（减dTTP的） 40微升的缺口转换酶 总体积200μL 孵育@ 16°CO / N。停止在70℃下加热反应10分钟。冰上冷却5分钟。 </li><li>通过添加以下的探针混合物，用乙醇沉淀DNA: 200μL的探测解决方案 48微升3 M醋酸钠 160，微升Cot1DNA（0.25微克/ UL） 1200μL100％的乙醇</li><li>储存在-80℃下为10分钟到O / N。 </li><li>自旋IŇ冷以12,000 xg离心20分钟。倾出乙醇。清洗1次，用70％的乙醇。旋前。倾出乙醇。空气干燥，直至乙醇蒸发。重悬在40μl的蒸馏水2 O的100纳克/微升的BAC DNA的最终浓度。 </li></ol>

<ol>
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没有利益冲突的声明。

染色体利差的准备是成功的复制时序分析的关键步骤这里描述。列入的秋水仙胺预​​处理步骤之前，低渗处理可能有助于在频率和扩频的有丝分裂细胞。我们通常会公开细胞，以colcemdi的1-3小时收获前，和使用秋水仙碱，终浓度为10微克/毫升。然而，列入秋水仙碱预处理步骤可能会改变G2的长度，因此可能会改变明显凝结染色体的复制时间和状态。 此过程可以应用?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>抗BrdU-FITC </td><td>罗氏微孔</td><td> 11202593001 MAB326F </td><td> 50微克/微升</td></tr><tr><td>尼克翻译工具包</td><td>雅培分子（Vysis） </td><td> 07J00-0001 </td><td></td></tr><tr><td>频谱橙色的dUTP </td><td>雅培分子（Vysis） </td><td> 02N33-050 </td><td></td></tr><tr><td> CEP </td><td>雅培分子（Vysis） </td><td>而异</td><td></td></tr><tr><td> LSI / WCP杂交的缓冲区</td><td>雅培分子（Vysis） </td><td> 06J67-011 </td><td></td></tr><tr><td> CEP杂交缓冲液</td><td>雅培分子（Vysis） </td><td> 07J36-001 </td><td></td></tr><tr><td>染色体排新界南总区</td><td> MetaSystems集团</td><td> D-14NN-050-TR </td><td></td></tr><tr><td>奥林巴斯BX61荧光显微镜</td><td>奥林巴斯</td><td> BX61TRF-1-5 </td><td></td></tr><tr><td>显微镜成像软件系统</td><td>应用成像</td><td> Cytovision 3.93.1 </td><td></td></tr><tr><td>数码相机</td><td>奥林巴斯</td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> 原位杂交食谱 甲酰胺解决方案 70％Formamide/2x SSC 35毫升甲酰胺（Sigma公司） 10毫升10倍SSC 5毫升D 2 H 2 0 pH值至7.0，用HCl（Sigma）的 *重要的是使用甲酰胺已被存储于-20℃下长时间室温下存放会产生甲酸和PH W生病太低。 50％Formamide/2x SSC 25毫升甲酰胺（Sigma）的 10毫升10倍SSC 15 ML DH 2 O pH值至7.0，用HCl（Sigma）的 20×SSC，4 L 702克氯化钠（Sigma公司） 358克柠檬酸钠（Sigma公司） DH 2 O定容 PN缓冲区[0.1中号NaP0 4 0.1％NP_40（Sigma公司）] 请的0.1M溶液每个磷酸钠（过滤器消毒和储存在500毫升的等分试样）。 的0.1M的NaH 2 PO 4，1升 13.8克的NaH 2 PO 4（Sigma公司）: DH 2 O定容 0.1 M的NaH 2 PO 4 1大号 </strONG&gt; 14.2克的NaH 2 PO 4（Sigma公司） DH 2 O量。 PN:0.1 M的pH值调整的Na 2 HPO 4 pH值至8.0 .1 M的NaH 2 PO 4。过滤消毒，加1毫升的NP-40。 PNM50毫升 1.25克非脂肪干奶（Sigma）的 25毫升PN缓冲液（配方以上） 混合15-20分钟，并不断搅拌。附带的2倍，在400×g离心10分钟。使用上清，确保不打扰沉淀牛奶中的蛋白质。 </td></tr></tbody></table>

chromosome replicating timing combined with fluorescent in situ hybridization

哺乳动物DNA复制的启动在染色体上的多个站点在不同的时间在S期，随着时间的复制程序。被认为是一个动态的过程受反应的表观遗传修饰的组织特异性和发展线索，规范的复制时间。但是，调节，当启动染色体上DNA复制的机制仍然知之甚少。同源染色体的复制同步，但有显着的例外情况。例如，在雌性哺乳动物细胞中的两个X染色体中的一个成为后期复制经历一个过程被称为X染色体失活1。除了这种延迟复制时间，估计要2-3小时，一个X染色体上，成为大多数基因转录沉默。此外，一个离散的顺式作用位点，被称为X染色体失活中心，调节此X染色体失活过程中，包括第Ë感应对整个失活的X染色体的复制时间延迟。此外，一些在肿瘤细胞和细胞中的染色体重排暴露于电离辐射，显示复制时间&gt; 3小时，影响了整个染色体2,3显着延迟。我们的实验室最近的工作表明，中断的离散顺式作用常染色体位点在极晚，影响了整个第4号染色体的复制型。额外的染色体工程的研究表明，某些染色体重排，影响了许多不同的染色体造成这种不正常的复制计时表型，这表明所有的哺乳动物染色体包含离散的顺式作用位点，控制适当的个体染色体的复制时间。 在这里，我们提出了一个方法的定量分析结合荧光原位杂交染色体复制时间。这方法允许在同一细胞同源染色体之间的复制时间的直接比较，并被改编6。此外，这种方法可以明确识别的染色体重排，与影响整个染色体的复制时间变化的。这种方法有优势，最近开发的高通量微阵列或不能区分同源基因上的重新排列和重排染色体的测序协议。此外，因为这里描述的方法的计算结果单细胞，它可以检测染色体重排，在一个群体中只有一小部分的细胞中存在的染色体复制定时变化。

人染色体6的复制定时分析的一个例子示出在图2中。细胞中含有ASAR6基因4，位于6q16.1删除，被暴露5小时的BrdU的有丝分裂细胞，收获和加工FISH染色体6油漆探针（Vysis）和BrdU掺入。请注意，有是一个显著差的的BrdU条带图案的2 6的之间，这是与一个延迟复制定时&gt; 2小时中的染色体6的[见4的复制定时绑扎6号染色体模式之一相一致之前删除。此外，有一个显着差异时BrdU掺?...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

染色体复制定时分析的定量方法进行说明。的方法，利用结合BrdU掺入荧光<em在原位</em&gt;，杂交（FISH），以评估哺乳动物染色体的复制定时。这种技术允许在同一细胞重新排列和未重排的染色体直接比较。

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

chromosome-replicating-timing-combined-with-fluorescent-situ

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

13.9K 次观看.  Oregon Health & Science University. 染色体复制定时分析的定量方法进行说明。的方法，利用结合BrdU掺入荧光

视频: Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

科研

遗传学

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

发育生物学

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

结合DNA-RNA荧光<em&gt;原位</em&gt;杂交（FISH）研究X染色体失活的分化雌​​性小鼠胚胎干细胞

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

生物学

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

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.

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

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.

快速荧光<em&gt;原位</em&gt;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

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

对于荧光DNA的简易方法<em&gt;原位</em&gt;杂交来压扁染色体

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

DNA晕片制备物的荧光原位杂交揭示整个染色体、端粒和基因位点

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, together known as its karyotype. Many chromosomal abnormalities are associated with disease. Each chromosome in a karyotype can be stained with a variety of dyes to give unique banding patterns. More recent techniques, including comparative genomic hybridization and fluorescence in situ hybridization (FISH), allow for detecting specific chromosomal features or abnormalities.This video will begin by examining the principles of these classical and modern cytogenetics techniques. This is followed by an examination of a general protocol for performing FISH. Finally, several examples of how karyotyping can be applied to various medical applications are presented.

结合荧光染色体复制时序

摘要

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此视频中的章节

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

通过多色荧光跨染色体畸变稳定的检测

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

结合DNA-RNA荧光

快速荧光

对于荧光DNA的简易方法

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

DNA晕片制备物的荧光原位杂交揭示整个染色体、端粒和基因位点

细胞遗传学