作者感谢概述的意见和艾伦·科克伦的实验室方法的发展作出贡献的过去和现在的成员。 LA是一个加拿大卫生研究所（CIHR）博士生奖学金和AJM研究院收件人，由弗雷泽，Monat和麦弗逊式事业奖的支持。支持这项工作是由一个从CIHR（赠款＃澳门-56974）的资助。

1。探针的制备<ol><li>精华1微克， 在体外转录载体，PKS（+），1小时9 POL 236nt Kpn1酶在37°C和运行上的线性DNA的琼脂糖凝胶产品。片段应该是约2500 bp的。 </li><li>切成片段，凝胶净化使用罗氏微洗脱凝胶提取试剂盒（ 表1中列出的所有试剂使用）。执行最终在30μL洗脱缓冲液洗脱。运行5μL琼脂糖凝胶电泳验证净化产品。 </li><li>混在体外转录反应使用罗氏辛RNA标记试剂盒和孵育2小时，在37°C（见下面的配方）。 </li></ol> 在体外转录反应： <table border="1"><tbody><tr><td>线性的DNA </td><td> 23μL </td></tr><tr><td> 1X辛RNA标记组合（罗氏） </td><td> 4μL </td></tr><tr><td> 1X茶nscription缓冲区</td><td> 8μL </td></tr><tr><td> 20U RNase的输出</td><td> 1μL </td></tr><tr><td> 80U的T7 RNA聚合酶</td><td> 4μL </td></tr><tr><td>总</td><td> 40μL </td></tr></tbody></table><ol start="4"><li>在37°C为15分钟，加入228的DNase I和孵育ü。 </li><li>停止反应，加入EDTA的pH值8.0至20毫米的终浓度。 </li><li>净化罗氏公司采用快速自旋列由生产厂家指定的反应。 </li><li> DEPC处理过的1/10体积的3M醋酸钠pH值5.2和2.5体积冰冷的95％乙醇沉淀净化探头。混合，并在-80°C孵育30分钟至过夜。 </li><li> 15分钟，在4°C离心15,000 xĞ探头。 </li><li>去除上清，在冰冷的70％乙醇洗沉淀。 15,000 xĞ再次离心5分钟在4°C。 </li><li>取出上清液，干燥沉淀。在50μL瓦特悬浮颗粒亚特（DNA酶/核糖核酸免费）含10üInvitrogen公司的核糖核酸酶输出。 </li><li>估计分光光度法探头（在260 nm的措施RNA）的浓度，并稀释至5 ng /μl的浓度。商店在短期使用，或-80°C下长期贮存于-20°C等分。它建议执行间的分析比较，以便保护这等份。 </li></ol> 2。细胞固定<ol><li>它最初是重要的种子将未经处理的，无菌的盖玻片的贴壁细胞，所以最终汇合后收获约70-80％。对于非贴壁细胞，生长正常，并准备收集时，孵育1小时，已与0.01％W / V聚 - L-赖氨酸（Sigma-Aldrich公司，公司）治疗的盖玻片。对于一个典型的12孔聚苯乙烯组织培养板（3.8厘米），15细胞（如HeLa细胞）镀在转染前24小时。非贴壁细胞可以像往常一样感染或转染，并允许广告这里固定3前的玻璃盖玻片。 </li><li>丢弃的媒体和双蒸馏水（DPBS）1分钟准备1X的磷酸盐缓冲液洗细胞。酸二乙酯（DEPC处理，Sigma-Aldrich公司，公司），1×PBS也可用于处理，但未经处理的PBS不能因为它可能含有核糖核酸酶。 </li><li>倒掉PBS和添加4％多聚甲醛，足以完全覆盖细胞。孵育15-20分钟。 </li><li>丢弃paraformaldeyhyde和用1X DPBS为1分钟的细胞。 </li><li>丢弃的DPBS和添加0.1 M甘氨酸（在1X DPBS解散）。孵育10分钟。 </li><li>丢弃的甘氨酸和用1X DPBS为1分钟，细胞。 </li><li>丢弃DPBS和添加0.2％的Triton-X的（在1X DPBS解散）。孵育5-10分钟。如果核仁或核包膜蛋白染色，通透与海卫不超过5分钟或蛋白定位X-100的将成为弥漫。 </li><li>丢弃的Triton X-100洗一次1X DPBS为1分钟。 </li><li>对于长期储存，在4°C PBS取代了4个月，用70％乙醇和商店。另外，洗一次1X DPBS，并着手对鱼类。 </li></ol> 3。 荧光原位杂交技术（FISH） <ol><li>如果盖玻片存储乙醇，取代1X DPBS乙醇和使细胞水化30分钟。补液可以一蹴而就，如果盖玻片储存在4°C </li><li> 1X 1分钟DPBS清洗盖玻片。 </li><li>准备幻灯片孵育的盖玻片上，照顾他们清理和标签。 </li><li>对于18毫米盖玻片，添加50μLDNA酶溶液（25单位/盖玻片，市售）的幻灯片。加盖玻片，请务必将它放在电池的侧面下来，并在室温下15分钟孵育。 </li><li> 1X 1分钟DPBS清洗盖玻片。 </li><li>添加50μL杂交组合每盖玻片（见下文）配方到幻灯片和孵育16-18Ĥ盖玻片细胞的一面向下，在42°C。孵化应在含50％甲酰胺，2X SSPE的组合（12.5毫升去离子甲酰胺，2.5毫升20X SSPE的，10毫升水）的托盘。 </li></ol>杂交组合（一盖玻片，50μL）： <table border="1"><tbody><tr><td> 库存量 </td><td> 材料（终浓度） </td></tr><tr><td> 25μL </td><td>甲酰胺，50％（去离子;任何源） </td></tr><tr><td> 5μL（10毫克/毫升） </td><td> tRNA的1毫克/毫升（Sigma-Aldrich公司，公司） </td></tr><tr><td> 5μL（20X） </td><td> SSPE的，2X </td></tr><tr><td> 5μL（50X） </td><td> denharts，5X </td></tr><tr><td> 0.125μL（5个） </td><td>核糖核酸酶输出</td></tr><tr><td> 5μL（5 ng /μl的25毫微克） </td><td>探测器</td></tr><tr><td> 5μL </td><td> H 2 O DEPC处理，作出最后的卷50μL </td></tr></tbody></table><ol start="7"><li>孵育细胞盖玻片一侧，在50μL50％甲酰胺（在1X DPBS稀释）15分钟42°C。 </li><li>洗2X SSPE的盖玻片两次孵化它们细胞的一面，每次5分钟，在50μL2X SSPE的（20X SSPE的在1X DPBS稀释）在42°C。 </li><li>在1分钟1X DPBS冲洗盖玻片。 </li></ol> 4。免疫荧光染色<ol><li>阻止细胞放置在50μL1X罗氏封闭液（在1X DPBS稀释10X罗氏阻塞解决方案）朝下30分钟的盖玻片。 </li><li>孵育1小时50μL主要抗体溶液中的盖玻片细胞面朝下，在37°C。应根据制造商的方向1X封闭液稀释的抗地高辛抗体。如果正在使用其他抗体染色的蛋白质，他们可能会在正确的浓度增加，所有使用的抗体是从不同的主机规格而得IES。 </li><li>洗为10分钟1X DPBS盖玻片。 </li><li>孵育1小时50μL二次抗体溶液中的盖玻片细胞面朝下，在37°C。的Alexa Fluor标记的抗体（Invitrogen公司）可以用来生成颜色的范围，并在1:500 1X阻塞的解决方案，1X DPBS。 </li><li>洗盖玻片两次为1X DPBS各10分钟。 </li><li>盖玻片细胞侧干滤纸纸。一定覆盖盖玻片，以避免暴露于光线和漂白。 </li><li>一旦所有的液体蒸发，安装到使用8μLImmunoMount的Thermo Scientific的（公司）的新鲜幻灯片盖玻片。轻轻敲击盖玻片，以消除任何气泡。 </li><li>应用指甲油盖玻片的边缘，以确保到位。 </li><li>显微技术的RNA和蛋白质的可视化也是这一技术成功的关键。使用显微镜上的设置，可以帮助或阻碍的可视化，因此它是关键在显微镜上的设置正确设置和从一个样本到另一个在一个给定的实验是一致的。光学和抗体组合进行详细的显微镜设置，请参见参考资料1,10。 </li></ol> 5。代表结果病毒RNA染色的例子可以看出，在图2。 HIV-1病毒RNA被视为整个显示大部分弥漫的细胞质，细胞质punctae虽然小的情况并不少见。可以看出，通过比较阳性细胞周围的细胞不显示荧光的特异性。在介绍中提到，HIV-1，缺乏监管的Rev蛋白产生RNA，这是保留在细胞核：这可以是一个明亮的信号在细胞核中的可视化，缺乏在细胞质中的RNA荧光信号。细胞蛋白的过度表达也能转移的HIV-1病毒RNA的分布。在图2 </strong&gt;中，在胞内囊泡运输有关的蛋白质的过度表达（例如，Rab7互动溶酶体蛋白（RILP）11或N-末期删除RILP（RILPN）的11，蛋白与动力蛋白运动功能的干扰[例如p50/dynamitin 1， 12]，干扰正常的稳定状态的病毒基因组RNA的定位，通过FISH分析确定RILP表达导致病毒基因组RNA的重新定位，以微管组织中心（选委）13。RILPN分散后期内涵体内细胞质由于无法绑定到动力蛋白复合运动14大亚基的动力蛋白马达和发布p50/dynamitin块胶合 P150后期内涵体，而且病毒基因组RNA和HIV-1结构蛋白的细胞外围1（ 图2）操纵的病毒基因组RNA的稳态本地化，感染力的一个关键因素病毒颗粒，将是最终发展疗法的关键。在我们手中，病毒RNA在细胞中出现，早在3小时后转染和感染10，并成为这12个小时的FISH技术很容易观察到。 <img alt="图1" src="/files/ftp_upload/4002/4002fig1.jpg" /> 图1。盖玻片上生长的鱼/ IF共同分析细胞的协议流程图 ，然后用甲醛固定。甘氨酸和Triton-X的治疗方法是进行细胞之前，无论是用于鱼/ IF或储存在70％的乙醇储存。细胞储存脱水水化1X DPBS（DEPC处理，PBS），然后再继续上鱼/ IF。细胞治疗用DNase我留下过夜杂交探针。一旦完成杂交，洗涤细胞与甲酰胺，以及与SSPE的，最终1X DPBS冲洗。阻断剂应用于细胞，其次是培养与主要抗体。洗涤后，二次抗体应用。两个1X DPBS最后清洗完成后盖玻片干燥和幻灯片上的成像的染色过程。 <img alt="图2" src="/files/ftp_upload/4002/4002fig2.jpg" /> 图2。代表鱼/ IF共同分析结果A）HIV-1转染HeLa细胞，并在某些情况下，结构，导致细胞蛋白表达（RILP，p50/DynamitinRILPΔN（氨基末端缺失突变体）） 。鱼/ IF共同分析：RNA被确定G3BP或LAMP1（红色）在绿色来区分。乙）的HIV-1的表达在HeLa，并在不同时间点收集。病毒RNA的表达被跟踪而绿色的细胞蛋白，核蛋白A1，在红染色。大小酒吧10微米。从参考文献1（一个选择面板数字; RILP，p50/Dynamitin，和RILP DELTAN）修改图像和17（B组）。

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+mimics+of+green+fluorescent+protein.">RNA mimics of green fluorescent protein.</a> Science. 333, 642-646 (2011).</li><li>Eckhardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SNAP-tagged+derivative+of+HIV-1--a+versatile+tool+to+study+virus-cell+interactions.">A SNAP-tagged derivative of HIV-1--a versatile tool to study virus-cell interactions.</a> PLoS One. 6, e22007 (2011).</li></ol>

没有利益冲突的声明。

鱼/ IF共同分析是一种可靠的方法，可视化现已提炼9,15,16细胞的病毒RNA。在过去的几年中，我们已经开发出一种RNA的染色成品的方法。这种技术可用于广泛的细胞类型的阵列探头是特定的靶RNA 17。通过与地高辛标记的探针，我们能够通过简单的染色可视化的RNA。当染色RNA和其他蛋白质相结合，共同分析鱼/ IF成为一个强大的工具来观察细胞结构和蛋白质/ RNA本地化。 特异性的RNA检测是相当高的。 图2说明了这一点。在一些原来的工作重点，对病毒基因组RNA的定位，建立鱼，缺乏牧师被困在细胞核内的病毒RNA 18。因为这，丰富的新病毒基因组RNA的本地化信息已获得使用的techniqu“é概述。由过度的细胞蛋白，并非所有的病毒RNA和人群定位的不同区域的细胞，可以强制。类似的表型的RILP表达，获得核蛋白A2的siRNA枯竭时在HIV-1表达13日 ，会导致RNA的明显积聚在选委。病毒基因组RNA可以减去高端马达蛋白，动力蛋白：p50/Dynamitin过度或击倒的动力蛋白重链1，释放病毒基因组RNA内域结果通过禁用蜂窝边缘推到细胞边缘（ 图2）。突变体的RILP，RILPΔN，不再结合的动力蛋白马达，分散内涵体进入细胞质（LAMP1标签），因为他们不再积极在juxtanuclear域本地化。这些结果指出，人口的HIV-1病毒基因组RNA，特别是塑料的概念，不同的HIV-1基因池RNA的识别作用在病毒复制周期有时存在。这些相同的概念已经确定了这个基因编码的蛋白质，加格19。 鱼/ IF合作与抗体选择和可用性分析的用途有局限性。小学和中学的抗体，其浓度的最佳组合优化需要时间来优化（ 见表1和2）。从杂交瘤细胞产生的抗体或由各大公司可以有不同任何地方，从1:2到1:2000，但一定要遵循的指导方针，以取得最佳效果的制造商提供的浓度。在其中的抗体产生的主机，还必须考虑下。由于跨物种识别（数据未显示），在高背景相结合的结果，也应避免在小学和中学的抗体混合绵羊与山羊抗体。对于网站的氟secondaRY抗体，可以用浓度为1:500几乎任何在一组的抗体。鱼/ IF共同分析，在本报告所述的其他缺点是它捕捉其在稳态位置的RNA，细胞已被固定后，使用多聚甲醛和洗衣粉（ 图1）和透。 然而，RNA在活细胞成像已经通过各种手段20-22，大多采用如GFP荧光蛋白标记的病毒RNA基因组的变化。然而，更多的手段来识别活细胞中的RNA本地化继续浮出水面。这些新方法涉及的标记RNA手段，菠菜23，24的SNAP或MTRIP 2。这些技术也遭受一些缺点，包括要求通透前增加基板，基必须设计标记的mRNA在显微镜检测的mRNA的细胞。事实上，主要ADVA在本报告所述的鱼类分析ntage在于在本地RNA导致生理上最相关的结果是不变的事实。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 18毫米盖玻片</td><td>厂商VWR </td><td> 48380 046 </td><td></td></tr><tr><td> 16％多聚甲醛</td><td>托德·贝克</td><td> S898-07 </td><td>到4％：在60°C溶解在500毫升1XDPBS 5滴氢氧化钠20克。关火，并储存在-20°C至7.2的pH值不超过70°C间！ </td></tr><tr><td> TRITON-X的</td><td> OmniPur </td><td> 9400 </td><td></td></tr><tr><td>微洗脱凝胶提取试剂盒</td><td>罗氏公司</td><td> D6294-02 </td><td></td></tr><tr><td>辛RNA的标签混合</td><td>罗氏公司</td><td> 11277073910 </td><td></td></tr><tr><td>转录的T7 RNA聚合酶</td><td> Invitrogen公司</td><td> 18033-019 </td&gt; <td></td></tr><tr><td>快速离心柱</td><td>罗氏公司</td><td> 11814427001 </td><td></td></tr><tr><td> DNA酶I </td><td> Invitrogen公司</td><td> 18047-019 </td><td></td></tr><tr><td> 1X DPBS </td><td> Gibco公司</td><td> 14190-250 </td><td></td></tr><tr><td>甲酰胺</td><td>电解二氧化锰</td><td> FX0420-8 </td><td></td></tr><tr><td>氨酰tRNA </td><td> Invitrogen公司</td><td> 15401-021 </td><td></td></tr><tr><td>核糖核酸酶输出</td><td> Invitrogen公司</td><td> 10777-019 </td><td></td></tr><tr><td>辛检测荧光抗体增强设置＃4（封闭液） </td><td>罗氏公司</td><td> 1768506 </td><td></td></tr><tr><td>的Alexa Fluor抗体</td><td> Invitrogen公司</td><td>见表2 </td><td></td></tr><tr><td>杂交炉</td><td>布克尔科学entific </td><td>型号24100 </td><td></td></tr><tr><td> microslides </td><td>厂商VWR </td><td> 48300-047 </td><td></td></tr><tr><td> immunomount </td><td> thermoscientific </td><td> 9990402 </td><td></td></tr></tbody></table> 表1特定的试剂和设备的鉴定。 <table border="1"><tbody><tr><td> 种抗地高辛抗体（稀释;公司，目录号） </td><td> 颜色 </td><td> 常用的Alexa Fluor（1:500） </td></tr><tr><td rowspan="3">鼠标（1:500;西格玛，B7405） </td><td>绿色</td><td>的Alexa Fluor 488驴抗小鼠（Invitrogen公司，A21202） </td></tr><tr><td>红色</td><td>的Alexa Fluor 594驴抗小鼠（Invitrogen公司，A21203） </td></tr><tr><td>蓝色</td><td>的Alexa Fluor 647驴抗小鼠（Invitrogen公司，A31571） </td></tr><tr><td rowspan="3">羊（1:200;罗氏，11376623） </td><td>绿色</td><td>的Alexa Fluor 488驴抗羊（Invitrogen公司，A11015） </td></tr><tr><td>红色</td><td>的Alexa Fluor 594驴抗羊（Invitrogen公司，A11016） </td></tr><tr><td>蓝色</td><td>的Alexa Fluor 647驴抗羊（Invitrogen公司，A21448） </td></tr></tbody></table> 表2。组合二次抗体和抗地高辛，列入目录的数量和浓度。

detection of viral rna by fluorescence in situ hybridization (fish)

病毒感染细胞引起正常细胞的功能，为病毒复制的能源和资源转移的具体变化。被征用病毒宿主细胞功能的许多方面，通常是由病毒基因产物的表达，招宿主细胞的蛋白质和机械。此外，病毒工程师具体膜细胞器或移动囊泡和马达蛋白标记的目标地区（ 从头感染期间，病毒增选的分子马达蛋白，针对核细胞后，病毒装配过程中，他们将劫持细胞在病毒组装的机器，将帮助）。据了解病毒如何，特别是那些RNA基因组，协调细胞内的蛋白质和RNA的组成部分贩运，以及他们如何实现在细胞的特定基因的传染性颗粒组装。 RNA的定位研究开始于早期的工作。发展中国家较低的真核生物EMbryos和神经细胞提供了重要的生物信息，同时还强调，在基因表达的级联编程的RNA本地化的重要性。在其他生物体和细胞系统的研究已经取得了类似的重要信息。病毒是寄生菌，必须利用宿主细胞进行复制。因此，关键是理解RNA病毒如何通过核孔的细胞核，细胞质和其最终目的地之一到子代病毒粒子1，直接从他们的RNA基因组。 鱼作为一个有用的工具，以确定病毒RNA的稳态本地化的变化。结合免疫荧光（IF）22分析，鱼/ IF共同分析将提供合作本地化的蛋白质与病毒RNA的信息3时。因此，这种分析提供了一个很好的起点，其他生化或生物物理测试RNA-蛋白质相互作用4,5测试以来共定位，本身是没有足够的证据是一定的交互。在研究病毒RNA的本地化，使用这样的方法，已获得丰富的信息，病毒和细胞的RNA贩运活动6。例如，HIV-1感染细胞的细胞核中产生的RNA，但只有翻译在细胞质中的RNA。当缺少一个关键病毒蛋白（修订版）7，病毒RNA的鱼已发现的病毒复制的块是由于保留的HIV-1病毒基因组RNA在细胞核8。 在这里，我们提出病毒基因组RNA 原位可视化分析方法。该方法使使用一个标记的RNA探针。这种探头的设计是相辅相成的病毒基因组RNA。在体外合成的反义RNA探针，与地高辛（DIG），修改核苷酸包括在体外 transcription反应。一旦探针杂交细胞中的靶mRNA，随后的抗体标记步骤（ 图1）将揭示本地化的mRNA以及蛋白质的利益时，鱼/ IF。

Watch this Scientific Journal Video about Detection of Viral RNA by Fluorescence in situ Hybridization FISH at JoVE.com

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

荧光<em&gt;原位</em&gt;杂交（FISH）方法被开发的可视化检测病毒基因组RNA，用荧光显微镜。探针与特异性病毒RNA，然后可以使用杂交和免疫荧光技术相结合确定。这种技术提供了识别病毒的RNA或DNA在稳态本地化的优势，提供对控制细胞内的病毒贩卖事件的信息。

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

detection-of-viral-rna-by-fluorescence-in-situ-hybridization-fish

Research

JoVE Journal

Biology

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

38.4K 次观看.  Sir Mortimer B. Davis Jewish General Hospital. 荧光原位杂交（FISH）方法被开发的可视化检测病毒基因组RNA，用荧光显微镜。探针与特异性病毒RNA，然后可以使用杂交和免疫荧光技术相结合确定。这种技术提供了识别病毒的RNA或DNA在稳态本地化的优势，提供对控制细胞内的病毒贩卖事件的信息。

视频: Detection of Viral RNA by Fluorescence in situ Hybridization FISH

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

科研

生物学

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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 ...

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 ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...