作者想感谢迈克尔·Alifrangis和乌拉Abildtrup，基因分型的寄生虫和克里斯蒂娜·霍尔姆优秀的技术援助。这项工作是由霍华德休斯医学研究所（授予55005511），，Lundbeck公司基金（批准R9-A840）和尼尔斯·玻尔基金会。

1。新选择受感染的红细胞的生成对于这个测定法，获得最好的结果是当使用新鲜选择培养表面表达的PfEMP1蛋白。在这个特殊的实验3D7 P.恶性疟原虫谱系选择使用特定的抗体，如前面描述的1。 第1天<ol><li>嘉实200μl的包装血细胞从P.恶性疟原虫培养含有2-5％的IE后期通过离心分离，在800×g离心8分钟，在室温下。 </li><li>血液沉淀重新悬浮在2毫升37°C预热0.75％明胶溶液在一个14毫升的无菌管中，并离开为15-20分钟，在37℃下，以允许未感染和环阶段IE泥沙。 </li><li>收获后期的IE， 即上层相，到一个新的14毫升的试管中，并用10ml的37℃的温水RBC清洗介质清洗两次。 </li><li>用50微升具体的反PfEMP稀释在2毫升37℃的温水RBC清洗媒体和无菌过滤器1抗体。 </li><li>灭菌的稀释抗血清在14毫升无菌试管中，并培育30分钟，37°C下缓慢搅拌混合水洗后期的IE。 </li><li>附带下降，在800×g离心8分钟，在室温下，并清洗两次，每次用10毫升37℃的温水RBC清洗介质。 </li><li>洗两次50μL的Dynabead蛋白的悬浮液，红细胞洗涤介质中使用DynaMaq-15磁铁。 </li><li>在300μl红细胞洗涤介质中重悬磁珠和他们洗过的后期IE。在37℃下孵育30分钟，温和搅拌。 </li><li>管转移到DynaMaq-15磁体。静置1-2分钟，取出所有的液体。 </li><li>重悬磁珠于4-5毫升的红细胞洗涤介质。管转移回上的磁铁，让它为1-2分钟前消除所有的液体。重复洗涤步骤一次。 </li><li>将洗过的小珠转移到一个25厘米2无菌培养fLASK含有200μl的新鲜堆积的红血细胞。添加5-6毫升Albumax媒体进入文化。存放过夜，在37℃下在5％CO 2的 </li></ol>第2天<ol start="12"><li>从文化当选定的的IE已经reinvaded的鲜红色的血细胞将所有的文化，一个14毫升的无菌试管中取出磁珠。将管到DynaMaq-15磁铁离开1-2分钟。然后，全部倒掉培养瓶中的液体倒回。 </li><li>尽可能为几个周期的5％至10％，直到原虫和寄生虫的文化是在ringstage。 </li><li> IE应通过FACS分析，以确保正确的表面表型， 即蛋白表达的任一PFD1235w and/orPF11_0008已得到的1。 </li></ol> 2。探针制备反义RNA探针产生的从最的可变区的PFD1235w和PF11_0008 变种&lt;/ em&gt;的基因，P.恶性疟原虫 3D7基因组DNA。通过PCR扩增的DNA，并克隆到载体转录的pSPT18或19，分别根据制造商的说明（Roche公司）。使用地高辛RNA标记试剂盒RNA标记或生物素混合，分别与地高辛（DIG）或生物素标记探针（580个碱基对（bp）和590 bp的长度）。我们证实了特异性的探针，通过Northern印迹分析和由单一标签FISH分析1。 3。薄涂片和固定在原位杂交中的寄生虫在此之前第1天所有的步骤被执行的无RNase的环境中，所有试剂无RNase的或预先用焦碳酸二乙酯（DEPC）或RNase扎普（Invitrogen公司）。载玻片和盖玻片应该用酒精清洗，以除去残留的制造油脂。重要的是要在各步骤之间没有任何暂停执行协议为了尽量减少核酸酶污染的风险。 <ol><li>薄血膜的标准传播方法3。使用油100倍的显微镜物镜，算上IE浏览器和IE浏览器中的百分比计算的文化。检查，寄生虫阶段是在近似的同步的，在环和早期滋养体阶段的IE周期。这些都是预先复制，单核阶段，表达var基因mRNA的和适合这种类型的单细胞FISH转录分析。 </li><li> 50微升的寄生虫文化传输，在原虫的5-10％，以1.5毫升的无RNase的Eppendorf管中。在室温下以2,000 rpm离心1分钟。 </li><li>取出介质，并加入50μlPBS DEPC处理过的水的pH值为7.2。轻轻搅动管。离心沉淀步骤重复两次以洗涤细胞不受媒体和细胞碎片。 </li><li>四好品质的薄涂片。对于每一个涂抹，使涂抹于一个11毫米直径的4以及幻灯片， 即四在总的载玻片上，在一个无RNA酶罩（ 关键步骤 ）。波简要地滑动到空气中晾干。车型三个额外的任何其他的无关基因的单探针染色阴性对照 - 酮滑动滑动，通过使用特定的探头，另一个滑动核糖核酸酶处理和三分之一滑动含有IE不表达感兴趣的基因。留在板凳上干10-20分钟的幻灯片。 </li><li>修复的薄膜，通过添加60微升固定溶液含有4％多聚甲醛和5％冰醋酸入井。在室温下静置10分钟，在板凳上。 </li><li>通过温和搅拌5分钟，在室温下，在2×SSPE缓冲液洗净幻灯片。多余的液体接触的孔中的细胞，用纸巾轻轻取出。 </li><li>量的幻灯片0.01％的胃蛋白酶在0.01 M HCl 2分钟，在37°C（一个非常关键的一步）。洗5分钟，在室温下2个亚急性硬化性全脑炎中的幻灯片。 </li><li>的RNase溶液稀释至最终浓度为10微克/毫升。量的负控制涂片用60微升的RNase溶液。在37°C孵育30分钟，然后洗5分钟，在室温下2个亚急性硬化性全脑炎中的幻灯片。 </li><li>立即着手在杂交步骤。 </li></ol> 4。杂交的RNA探针固定滑轨第1天<ol><li>准备杂交室，作为温度感应器100 HC4的或空的移液管尖中放入干净的烘箱中，通过喷洒用RNase扎普和擦拭清洁用薄纸等。填充杂交室或用DEPC处理过的水，以使确保幻灯片不会干出期间杂交的框的底部的通道。关闭室和预热至48°C。 </li><li>稀释到杂交溶液至终浓度为12纳克/微升探针。变性的探针溶液在65℃的加热块中的5分钟。立即冰上冷却。 </li><li>简单地说晾干后2个亚急性硬化性全脑炎洗的幻灯片。孔周围用纸巾小心地除去过量的液体。 </li><li>打开杂交室，和室中的幻灯片。应用的总体积为60微升每孔中的一个或两个探针。一个无RNA酶的盖玻片，用无菌镊子小心地覆盖液滴。 </li><li>关闭腔，并在夜间至少​​16小时，在48°C杂交的幻灯片。 </li></ol> 5。抗体共轭荧光扩增第2天下面的步骤是基于TSA Plus荧光调色板的系统从Perkin Elmer。所有的步骤都在室温下进行。 <ol><li>洗净幻灯片的3倍TN​​T缓冲轻轻搅拌5分钟。 </li><li>井座TNB缓冲液150μl的30分钟。 </li><li>稀释的HRP共轭的α-DIG抗体1:100，HRP共轭的α-生物素抗体的比例在TNB的缓冲液中，在TNB缓冲区中的比率为1:500。用纸巾从幻灯片中删除任何多余的TNB缓冲。同时检测两个转录时，这两种抗体可以去一次。应用的抗体TNB的溶液，每孔加入100μl的总量，并小心地用盖玻片覆盖。孵育的幻灯片2小时。 </li><li>取出盖玻片仔细。洗净的幻灯片在TNT缓冲液3次，每次5分钟。 </li><li>在1:500的比例稀释的FITC-荧光团中的酪胺的扩增试剂，和应用的解决方案，每个孔中加入100μl。井用盖玻片覆盖，孵育12分钟。 </li><li>小心取出盖玻片，并用幻灯片3次，轻轻搅拌5分钟，TNT缓冲。 </li><li>在1:500的比例稀释Cyan3荧光团中的酪胺的扩增试剂。加入100μl每该溶液。井用盖玻片覆盖，孵育12分钟。 </li><li>取下盖子滑落仔细阅读，然后快速冲洗幻灯片，沉浸在TNT缓冲。 </li><li>幻灯片洗净3次，用TNT缓冲液5分钟。 </li><li>迅速在DEPC处理过的水浸泡幻灯片。 </li><li>离开幻灯片风干。安装防褪色试剂DAPI幻灯片。密封用指甲油的盖玻片的角部。 </li><li>现在准备用于显微镜的幻灯片。保持覆盖铝箔和存储的幻灯片，在4°C，如果实验是要完成的第二天。一个完整的密封的侧面的滑动，可以进行24小时后，施加的抗褪色试剂。这将允许被成像在幻灯片最多为一个星期后的协议已完成。 </li></ol> 6。杂交探针的可视化<ol><li>打开显微镜上。一个标准的免疫荧光显微镜，够ient这种实验，但如果你有机会到激光共聚焦显微镜，你也可以使用这个3D图像或获得视频染色细胞。允许显微镜热身15-30分钟。接通计算机和软件。 </li><li>质量检查的染色，免疫荧光显微镜。选择一个点，其中包含一些寄生虫。 </li><li>手动调整每个激光器的增益。调整的像素停留4-6米-1 s -1和512×512象素的步骤。 </li><li>测试图片使用框架拉姆达。调整激光的增益。 </li><li>以最低的20个好照片的荧光单细胞。需要至少有2-5个图片一个字段包含5-20寄生虫的阳性寄生虫的百分比，以获得一个公平的概述。 </li></ol>

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没有利益冲突的声明。

RNA FISH分析，如Northern印迹和RT-PCR的方法，允许特定的mRNA转录在单细胞水平上的歧视。这使得有可能区分转录活性和非活动单元，在这个例子中，P.恶性疟原虫在人体红血细胞内寄生原虫。这种全细胞观察往往是必要的，并可能解开重要的和新颖的转录模式1。 虽然其他RNA FISH的方法已被描述4-10，出现了一个需要发展的一个新的和精致的协议，用于研究?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>醋酸</td><td>适马/爱秩序</td><td> 338826-100ML </td><td></td></tr><tr><td> Albumax媒体：RPMI 1640谷氨酰胺的解决方案</td><td>龙沙</td><td> BE12-115F </td><td> 500毫升RPMI 1640 5毫升谷氨酰胺解决方案</td></tr><tr><td> Albumax媒体：硫酸庆大霉素Albumax的解决方案</td><td>龙沙</td><td> BE02-012E </td><td> 2.5毫升硫酸庆大霉素 50毫升Albumax解决方案</td></tr><tr><td> Albumax解决方案：次黄嘌呤</td><td> Sigma-Aldrich公司</td><td> H9377 </td><td> 0.8克次黄嘌呤</td></tr><tr><td> Albumax溶液：AlbuMAX II </td><td>我nvitrogen </td><td> 11021-037 </td><td> 200克Albumax </td></tr><tr><td> Albumax解决方案：RPMI 1640 </td><td>龙沙</td><td> BE12-115F </td><td> 4升RPMI 1640 与磁铁的最大溶解。 50°C。过滤消毒，分装储存在-20°C。 </td></tr><tr><td> AMBERLITE </td><td>西格玛</td><td> A5710-110G </td><td>去离子甲酰胺树脂。 </td></tr><tr><td>山羊多克隆抗生物素过氧化物酶结合物</td><td> Calbiochem公司</td><td> 203206 </td><td></td></tr><tr><td>抗地高辛抗体</td><td>诺伟司生物制品</td><td> NB100-41330 </td><td></td></tr><tr><td>防褪色试剂DAPI </td><td> Invitrogen公司</td><td> P36931 </td><td>安装媒体24 h后固化完全密封的滑动。 </td></tr><tr><td>生物素RNA标记液</td><td&gt;罗氏</td><td> 11685597910 </td><td></td></tr><tr><td>相机数码</td><td>尼康数码视线DC-F11 </td><td></td><td></td></tr><tr><td>盖玻片</td><td>门泽尔 - 格拉泽</td><td> 631-1570 </td><td></td></tr><tr><td>培养瓶中，25厘米Nunclon表面</td><td> Nunc公司</td><td> 156340 </td><td></td></tr><tr><td> DEPC </td><td> Fluka公司/适马</td><td> 32490-100ML </td><td>在1000毫升去&lt;br/&gt;离子水DEPC1毫升。搅拌棒和搅拌12小时。高压灭菌30分钟。 </td></tr><tr><td>数码相机</td><td>尼康数码视线DC-F11 </td><td></td><td></td></tr><tr><td>磁珠蛋白A </td><td> Invitrogen公司</td><td> 10002D </td><td></td></tr><tr><td> DynaMaq-15磁铁</td><td> Invitrogen公司</td><td> 123-01D </td><td></td></tr><tr><td>甲酰胺bioultra 99％ </td><td> Fluka公司/ SIGMA </td><td> 47671-1L-F </td><td>去离子甲酰胺：5克每100毫升甲酰胺中的离子交换树脂。搅拌30分钟。通过Whatman滤纸过滤。 </td></tr><tr><td> 明胶75％的解决方案：明胶</td><td> Sigma-Aldrich公司</td><td> G2500 </td><td> 3.75克鱼胶片在500毫升的RPMI 1640。加热至56°C溶解。 56°C时，过滤消毒以等分试样在-20℃下储存。 </td></tr><tr><td> 明胶0，75％的溶液：RPMI 1640培养液</td><td>龙沙</td><td> BE12-115F </td><td> 3.75克鱼胶片在500毫升的RPMI 1640。加热至56°C溶解。 56°C时，过滤消毒以等分试样在-20℃下储存。 </td></tr><tr><td> 谷氨酰胺的解决方案：L-谷氨酰胺</td><td> Sigma-Aldrich公司</td><td> G3126 </td><td>在500毫升0.9％的氯化钠14.6克L谷氨酰胺。溶解，滤波器sterilize分装并储存在-20°C。 </td></tr><tr><td> 谷氨酰胺的解决方案：盐酸</td><td>西格玛</td><td> H1758 </td><td>在500毫升0.9％的氯化钠14.6克L谷氨酰胺。溶解，过滤消毒，分装储存在-20°C。 </td></tr><tr><td> 杂交液：甲酰胺生物超99％SSC 20倍</td><td> Fluka公司/适马</td><td> 47671-1L-F </td><td>共有20毫升，保持冷冻分装在-20°C 10毫升去离子甲酰胺</td></tr><tr><td> 杂交液：登哈特的50倍的浓缩</td><td>西格玛</td><td> D2532 </td><td> 5毫升20xSSC </td></tr><tr><td> 杂交的解决方案：的酵母tRNARoche阻断剂</td><td>西格玛</td><td> R-6750 </td><td> 2毫升50倍登哈特 250微升20毫克每毫升酵母tRNA </td></tr><tr><td><s豪华型杂交液：鲑鱼精子DNA </td><td> Fluka公司/适马</td><td> 31149-106GF </td><td> 0.4克罗氏阻断剂 1毫升10毫克每毫升鲑鱼精子DNA（严重：在96℃下5分钟，然后加入到杂交溶液的变性鲑鱼spermDNA） </td></tr><tr><td>杂化温度感应器100 HC4 </td><td> Quantifoil仪器公司</td><td> 1004-0011 </td><td>通过杂交烤箱和DEPC水填充的无RNA酶的杂交室可以被替换。 </td></tr><tr><td>浸油UV透明的荧光自由</td><td>西格玛</td><td> 10976-1EA </td><td></td></tr><tr><td>免疫荧光共聚焦显微镜</td><td>尼康D-Eclipse的TE2000C </td><td></td><td></td></tr><tr><td> Nailpolish </td><td>可在任何药店</td><td></td><td></td></tr><tr><td> PBS 20倍：氯化钠 氯化钾 的Na 2 HPO 4×2H 2 O KH 2 PO 4 DEPC去离子H 2 O </td><td></td><td></td><td> ajust pH值至7.4 160克 4克 23克 4克 1升</td></tr><tr><td>多聚甲醛4％ </td><td> Fluka公司/ CHEMIKA </td><td> 76240 </td><td> 4克PFA 80毫升PBS / DEPC。加热到65°C，直到PFA溶解。加入20毫升PBS中，使溶液冷却。将pH调节至7.4。过滤器。分装存放在-20°C </td></tr><tr><td>多聚甲醛4％/酸醋酸5％ </td><td></td><td></td><td> 950微升多聚甲醛+ 50μL醋酸</td></tr><tr><td>胃蛋白酶</td><td>适马/爱秩序</td><td> P7000 </td><td></td></tr><tr><td>过氧化物酶的共轭反生物素</td><td> Calbiochem公司</td><td> 203206 </td><td></td></tr><tr><td> 红细胞洗涤介质：RPMI 1640谷氨酰胺解决方案</td><td>龙沙</td><td> BE12-115F </td><td> 500毫升RPMI 16405毫升谷氨酰胺解决方案</td></tr><tr><td> 红细胞洗涤介质：硫酸庆大霉素</td><td>龙沙</td><td> BE02-012E </td><td> 2.5毫升硫酸庆大霉素</td></tr><tr><td>核糖核酸酶</td><td>适马/爱秩序</td><td></td><td>制作10毫克/毫升原液。 </td></tr><tr><td>无RNA酶的1.5 ml管</td><td> Ambion公司</td><td> AM12450 </td><td></td></tr><tr><td>核糖核酸酶扎普</td><td> Ambion公司</td><td> AM9780 </td><td></td></tr><tr><td>幻灯片4口井11毫米</td><td> Thermo Scientific的</td><td> MENZXER306W </td><td></td></tr><tr><td> SSC 20倍：氯化钠</td><td> SIGMA /铝drich </td><td> S9625 </td><td> 3 M氯化钠（175克/升）的0.3M的Na 3柠檬酸x高2 O（88克/升），用1M HCl调节pH至7.0 </td></tr><tr><td> SSC 20倍：柠檬酸钠脱水</td><td>适马/爱秩序</td><td> W302600 </td><td> 3 M氯化钠（175克/升）的0.3M的Na 3柠檬酸x高2 O（88克/升），用1M HCl调节pH至7.0 </td></tr><tr><td> 亚急性硬化性全脑炎20倍：氯化钠</td><td>适马/爱秩序</td><td> S9625 </td><td> 175.3克氯化钠</td></tr><tr><td> 亚急性硬化性全脑炎20倍的NaH 2 PO 4 </td><td>适马/爱秩序</td><td> S0751 </td><td> 27.6克的NaH 2 PO 4。 </td></tr><tr><td> SSPE 20倍：4 EDTA粉</td><td></td><td></td><td> 9.4克EDTA粉 加入DEPC水，调节pH值7.4。汽车劈好20分钟</td></tr><tr><td> TNB缓冲：TNT缓冲液</td><td></td><td></td><td> 10毫升TNT缓冲</td></tr><tr><td> TNB缓冲液：封闭试剂</td><td></td><td></td><td> 0.05克阻断剂 要封闭试剂溶解，溶液加热到60℃，在搅拌下的1小时。贮存于-20℃下（来源于Perkin Elmer公司TSA协议）。 </td></tr><tr><td> TNT缓冲液：Tris / HCl中</td><td>西格玛</td><td> T1503 </td><td> 1M的Tris / HCl，pH值8.0 </td></tr><tr><td> TNT缓冲液：氯化钠</td><td> Sigma Aldrich公司</td><td> S9625 </td><td>百毫升 </td></tr><tr><td> TNT缓冲液：吐温20 </td><td>西格玛</td><td> 93773 </td><td> 5 M氯化钠30毫升 1毫升DEPC蒸馏水2 O869毫升 调节pH至7.5，在室温下。 （来源于Perkin Elmer公司TSA协议）。 </td></tr><tr><td> TSA加Cyanine3 /荧光系统</td><td> Perkin Elmer公司</td><td> NEL753000IKT </td><td>在实验开始前，请仔细阅读该协议从TSA Plus荧光调色板的系统。 </td></tr><tr><td>管14毫升无菌</td><td> ALMECO - CM LAB APS </td><td> 91016 </td><td></td></tr></tbody></table>

analysis of single-cell gene transcription by rna fluorescent in situ hybridization (fish)

恶性疟原虫感染的红细胞（IE）在疟疾感染的人脐静脉内皮受体介导的黏附表达的PfEMP1 由 var基因编码的蛋白变异。 单倍体P.恶性疟原虫基因组中藏着约60个不同的变种基因，其中只有一个被认为是转录每一次细胞在血液的感染阶段。如何实现这种相互排斥的变种基因的转录调控还不清楚，因为是个人var基因的鉴定或小组var基因与不同的受体和后果的差分结合的临床结果P.恶性疟原虫感染。近日，被称为相互排斥的转录模式提出了质疑，根据个人P.转录分析恶性疟原虫转录鉴定单INF的ected红细胞细胞利用RNA 荧光原位杂交（FISH）分析VAR的寄生虫P.单个核基因转录恶性疟原虫即：1。 在这里，我们提出了一个详细的协议进行分析变种基因的转录的RNA-FISH方法在单核的P.恶性疟原虫感染人类的红细胞。该方法是基于对使用地高辛和生物素标记的反义RNA探针使用的TSA加荧光调色板系统2（Perkin Elmer公司制），微观分析和新鲜选择P.恶性 IE浏览器。 原位杂交法可以用来监测转录的各种基因表达和调节过程中的不同阶段的P.恶性疟原虫生命周期和适应其他疟疾寄生虫物种和其他生物体和细胞类型。

图1示出的主要步骤的流程图，和持续时间的RNA FISH方法。 一系列有代表性的图像，保存差染mRNA的FISH实验采用单P.疟原虫 IE浏览器在图2中所示。细胞内的原虫有一个小的（1-1.5微米直径）的原子核，用DAPI染成蓝色。二十四小时后，红细胞的入侵P.恶性疟原虫裂殖过程中， 即细胞核分裂，就开始了。优化FISH检测的变种基因转...

Watch this Scientific Journal Video about Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH at JoVE.com

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

荧<em在原位</em&gt;杂交（FISH），以确定在单个细胞中的mRNA转录如同时转录中的一个以上的成员的允许多基因活性分析<em&gt; VAR</em&gt;多基因家族<em&gt;恶性疟原虫</em&gt;受感染的红细胞<sup&gt; 1</sup&gt;。该技术的适应性和可用于不同类型的基因，细胞和有机体。

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 ...

analysis-single-cell-gene-transcription-rna-fluorescent-situ

Research

JoVE Journal

Biology

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

15.3K 次观看.  University of Copenhagen. 荧

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

科研

生物学

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 ...

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 ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

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 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

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 ...

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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...