我们感谢的萨沙赖希曼和多米尼克Santiard男爵为他们的帮助，在编辑RNA纯化协议。

1。日报:过程来恢复从外科块标本在实验室<ol><li>从快递公司的进口合同。 </li><li>填写10（或25）的运输形式实验室实验室的联络人（电话号码和电子邮件地址）的邮政地址。 </li><li>准备10（或25）样品编号从1到10（或25）的形式。这些形式包括专用空间通知）匿名鉴定的病人，B）的手术日期和c）任何额外的言论外科医生想补充。准备10（或25）软垫信封（150 x 210毫米）。 </li><li>用无RNA酶试剂25毫升（或65）6 M盐酸胍酸二乙酯（DEPC）处理过的H 2 O（GHCL）。 </li><li>填写10或25号5毫升无菌聚乙烯圆底管GHCL溶液2.4毫升。 </li><li>二手氩气，以填补这些管子的顶端部分，比按TI机已上的盖，以防止存储在过去几年，在手术机柜GHCL溶液氧化。 </li><li>介绍，每5毫升管50毫升无菌聚丙烯锥形筒底部用螺丝帽。使用一块干净的纸巾，保持管5毫升到50毫升管，管关闭。 </li><li>将50毫升管聚苯乙烯机架和机架到一个纸箱软垫信封，航运形式，样品形式。 </li><li>粘贴的纸板箱的盖子里面的说明（参见:1.12-1.19）​​，以方便他们阅读。 </li><li>通过邮件发送的纸箱在医院的联络人。 </li></ol>在医院<ol start="11"><li>把纸箱从手术室手术柜。注意注意:如果过程涉及免疫系统受损的患者，纸板不应该被带到手术室。 </li><li>在手术过程中，将视网膜SPECIM连接到编号为5毫升无菌聚丙烯填充GHCL的解决方案。关闭严密管。 </li><li>返回管简略。 </li><li>将试管的血管摇臂10分钟。 </li><li>填写随附的样品编号的形式。 </li><li>报告到相应编号的50毫升管的识别号码。 </li><li>介绍与手术标本管5毫升到50毫升管，取代纸巾，并拧紧管。 </li><li>引入管和填充样品形式把它在一个随附软垫信封。关闭它。 </li><li>拿起致电快递公司。 </li></ol> 2。 RNA纯化在实验室<ol><li>均质GHCL溶液用均化1分钟的聚丙烯与5毫升管中的试样移动的同时向上和向下的管子。 </li><li>加入270微升的2M乙酸钾pH值为5.0。管放置在振动筛上，在其大力摇晃10分钟水平位置（420分钟-1）。 </li><li>在5,000 rpm（6,500 XG）在20℃下离心10分钟</li><li>顺利地吸出可溶部分，而不会干扰沉淀。 </li><li>转移到一个14毫升的无菌试管。 </li><li>添加5.3毫升100毫米的Tris pH值8.0，N-月桂酰肌氨酸1％。 </li><li>添加3.2克氯化铯（CSCL），涡旋混合管。 </li><li>在一个无菌的11毫升的聚异分的离心管中，添加1.8毫升的氯化铯/乙二胺四乙酸（EDTA）。 </li><li>用10毫升无菌巴斯德移液管，管的边缘上缓慢滑动，以避免干扰的密度胶垫RNA溶液转移到1.8毫升氯化铯/ EDTA。 </li><li>入转子，将反应管（第二管中，如有必要，视网膜无缓冲区）。 </li><li> （225,000 XG）在32000转离心24小时，在20°C </li><li>用无菌巴斯德吸管取出优越的解决方案的一部分，并丢弃。 </li><li>逐步移除，同时检查时刻与第二无菌巴斯德吸管DNA（粘性）时吸气，并将其丢弃。 </li><li>小心不要释放RNA沉淀与第三无菌巴斯德吸管中取出剩余的溶液。 </li><li>第试管底部，用手术刀火焰消毒，然后把管倒挂一个无菌的目光上的剩余部分。 </li><li>扭转管和160微升GHCL微妙冲洗。 </li><li>让制粒干燥10分钟。 </li><li>将沉淀重悬于150微升（10mM的Tris pH值7.5 - 1mM EDTA的 - 0.1％SDS）。 </li><li>将溶液转移到2毫升无菌离心管中，然后收获剩余的30微升（10毫米的Tris pH值7.5 - 1毫米EDTA - 0.1％SDS）沉淀。 </li><li>加入150μl中（10mM Tris pH值7.5 - 1mM EDTA）中。 </li><li>加入30μl的3M醋酸钠pH值为5.0，涡管。 </li><li>加入900μl的100％乙醇（-20℃），涡管。 </li><li>将融化的冰管30分钟。 </li><li>岑trifuge管30分钟，以15,000 rpm于4℃。 </li><li>吸微妙可溶部分，并丢弃。 </li><li>加入500μl70％乙醇（RT），涡旋管。 </li><li>离心管20分钟，以15,000 rpm（18,000 XG）在4℃下</li><li>重复漂洗步骤（70％乙醇）。 </li><li>短暂离心，消除剩余的乙醇与P200的移液器。 </li><li>让沉淀空气干燥10分钟。 </li><li>悬浮颗粒在50μLDEPC处理的H 2 O </li><li>大力涡旋混合。 </li><li>孵育15分钟，在45℃的水浴中。 </li></ol> 3。 RNA通过凝胶电泳分析<ol><li>倒入琼脂糖凝胶化学罩内。 </li><li>在无菌1.5毫升离心管中，加入2μl的RNA进行分析和样品制备缓冲液6.4微升。 </li><li>在第二个1.5毫升试管中，加入3μl的RNA标准品和样品制备缓冲液9.6微升。 </li><li>加热管15分钟，在65℃下，把它们放在冰上。 </li><li>无染料的RNA样品管和1微升的EB样缓冲液与染料中的RNA标准品管中加入1μl溴化乙锭（EB）的上样缓冲液。 </li><li>运行的凝胶在80 - 100伏的专家在运行缓冲液的化学罩，直到其中的染料溴酚蓝到达凝胶底部的2/3的。 </li><li>冲洗凝胶，用250毫升去DEPC处理2X SSC两次15分钟。 </li><li>在紫外光照射下，以数字化图像。 </li><li>计算上带（23S rRNA的）和低频（16S rRNA基因）之间的比率。 </li></ol> 4。最终纯化的RNA <ol><li>的RNA样品的体积调整至100μlDEPC处理过的H 2 O（如有必要）。 </li><li>添加100微升酸酚（1毫升苯酚DEPC处理的H 2 O + 130微升50毫米醋酸钠，pH值5.2），涡大力饱和。" </li><li> （15,000 XG），在室温下以13000rpm离心10分钟。 </li><li> Recoveŗ仔细和转移为一种新型无菌1.5毫升离心管中水相（上期）。 </li><li>加入10μl的3M醋酸钠，pH值5.2和250μl的100％乙醇（-20℃）。 </li><li>孵育管30分钟到融化的冰。 </li><li>离心管30分钟，每分钟14000转（18,000 XG）在4℃下</li><li>仔细吸可溶部分，并丢弃。 </li><li>添加500μl的70％乙醇（室温）下，涡管。 </li><li>离心管20分钟，每分钟14000转（18,000 XG）在4℃下</li><li>重复漂洗步骤（70％乙醇）。 </li><li>短暂离心，消除剩余的乙醇与P200的移液器。 </li><li>让空气干燥沉淀10分钟。 </li><li>到50微升DEPC处理过的H 2 O 中重悬沉淀</li><li>管15分钟，在45℃下孵育</li><li> 2微升RNA样品上，测量在260 nm和280nm处的吸光率（Abs）。的比例计算Abs260/Abs280。 </li> &lt;LI&gt;商店的RNA样品在-80°C（稳定数年）。 </li></ol> 5。解决方案和清洁程序<ol><li> 2 M醋酸钾，pH值5.0:溶解19.6醋酸钾为70毫升DEPC处理的H 2 O到用1M NaOH中浸泡15分钟，然后用DEPC处理过的H 2 O冲洗5次清洁的pH计探针用醋酸调节pH值至5.0，然后调整音量至100 mL DEPC处理的H 2 O </li><li> ，pH值8.0的Tris-HCl 100mM的N-月桂酰肌氨酸1％:将2克N-月桂酰肌氨酸（下罩）的0.5M Tris-碱pH值8.0〜40毫升。 DEPC处理的H 2 O调节音量至200毫升）中海集运/ EDTA:中海集运96克50毫升10毫米EDTA pH值7.5编写DEPC处理的H 2 O高压灭菌，并调整体积至100毫升，用DEPC处理过的H 2 O的检查pH值&lt;7.5。 </li><li>琼脂糖凝胶1％:将1克琼脂糖在62毫升DEPC处理的H 2 O在微波炉中煮。添加18毫升为12.3 M甲醛，20毫升5×MOPS。倒入化学罩内。 </li><li>样品制备缓冲器:要准备即席。混合化学罩8微升5×MOPS，16微升12.3中号甲醛，甲酰胺和40μl内。 </li><li> EB样缓冲液（不含染料）:要准备的即席。入20μl的80％的甘油，用DEPC处理过的H 2 O制备，加入4微升10毫克/毫升的溴化3,8 -二氨基-5 -乙基-6 -苯基菲啶溴化</li><li> EB样缓冲液（染料）:要准备的即席。入10μl的80％甘油中含有0.25％的红外谱图严重混叠氰DEPC-处理过的H 2 O，0.25％溴酚蓝，加入2μl10毫克/毫升的溴化3,8 -二氨基-5 -乙基-6 -苯基菲啶溴化</li><li>电泳缓冲液:60毫升5倍MOPS，增加12.3中号甲醛54毫升和186毫升DEPC处理的H 2 O </li><li>用DEPC处理过的H 2 O:0.1％体积/体积焦碳酸二乙酯2小时孵育蒸馏水中，在37℃，同时搅拌。高压灭菌消毒。 </li><li>清洁清洗液中加入25毫升（RBS）均质:1升的DEPC处理的H 2 O，并浸入轴搅拌1分钟。孵育2小时，在60℃下彻底冲洗成铝DEPC处理的H 2 O将仪器放入仪器箱。包装盒。高压灭菌消毒。 </li><li>清洁电泳设备:清洗装置，托盘和15井梳。孵育15分钟，在3％的过氧化氢。用100％乙醇冲洗一次。空气干燥。 </li><li>清洁玻璃菜:清洁玻璃板菜RBS 2％所有的夜晚，然后在200℃灭菌前用蒸馏水冲洗</li></ol>

<ol>
	<li>Mitry, D., Charteris, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+rhegmatogenous+retinal+detachment:+geographical+variation+and+clinical+associations.">The epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations.</a> The British Journal of Ophthalmology. 94 (6), 678 (2010).</li><li>Green, R. E., Krause, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+draft+sequence+of+the+Neandertal+genome.">A draft sequence of the Neandertal genome.</a> Science. 328 (5979), 710 (2010).</li><li>Glisin, V., Crkvenjakov, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleic+acid+isolated+by+cesium+chloride+centrifugation.">Ribonucleic acid isolated by cesium chloride centrifugation.</a> Biochemistry. 13 (12), 2633 (1974).</li><li>Van Gelder, R. N., von Zastrow, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (5), 1663 (1990).</li><li>Cavalieri, L. F., Yamaura, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+tRNAs+as+inhibitors+of+viral+reverse+transcription+in+vitro.">coli tRNAs as inhibitors of viral reverse transcription in vitro.</a> Nucleic Acids Research. 2 (12), 2315 (1975).</li><li>Sawadogo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+inhibition+of+yeast+RNA+polymerases+A+and+B+by+tRNA+and+alpha-amanitin.">On the inhibition of yeast RNA polymerases A and B by tRNA and alpha-amanitin.</a> Biochemical and biophysical research communications. 98 (1), 261 (1981).</li><li>Delyfer, M. N., Raffelsberger, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+analysis+of+human+retinal+detachment+reveals+both+inflammatory+response+and+photoreceptor+death.">Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.</a> PloS ONE. 6 (12), e28791 (2011).</li><li>Kalathur, R. K., Gagniere, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RETINOBASE:+a+web+database,+data+mining+and+analysis+platform+for+gene+expression+data+on+retina.">RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina.</a> BMC Genomics. 9, 208 (2008).</li><li>Nakazawa, T., Hisatomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+chemoattractant+protein+1+mediates+retinal+detachment-induced+photoreceptor+apoptosis.">Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (7), 2425 (2007).</li><li>Leveillard, T., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod-derived+cone+viability+factor+for+treating+blinding+diseases:+from+clinic+to+redox+signaling.">Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling.</a> Science Translational Medicine. 2 (26), 26ps16 (2010).</li><li>Chalmel, F., Leveillard, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod-derived+Cone+Viability+Factor-2+is+a+novel+bifunctional-thioredoxin-like+protein+with+therapeutic+potential.">Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential.</a> BMC Molecular Biology. 8, 74 (2007).</li></ol>

什么都没有透露。

从手术块组织恢复发展的过程一直视网膜脱离的转录组分析的关键。人们应该注意到，这种类型的手术眼科医生经营，很少有时间参加一个生物研究计划，当他们在紧急情况和实践。此retinectomy也随机在每个服务，让简单的​​方法来达到统计上的数字是与网络。在这样的网络中，标准化组织集合是必不可少的成功的生物分析。通过提供的材料，非常容易使用和明确的指示，可在室温下储存在手?...

视网膜脱离（RD）治疗的主要目标是找到一种方法，以限制视细胞损伤和视网膜炎症产生的光感受器的视网膜色素上皮细胞的分离。道期间，视网膜色素上皮细胞被激活，迁移，脱分化，增殖的表面脱离的视网膜，施加收缩力，导致并发症。转录组分析的RD是一种识别靶基因表达修改后RD，因此未来的治疗性分子，可以提高最终视觉效果与手术的组合。脱氧核糖核酸（DNA），核糖核酸（RNA）是不稳定的，这是众所周知的，后者被广泛用于遗传研究的，其稳定性允许的来自古生物学样品中的超过30,000岁2尼安德特人基因组的测序。转录成信使RNA的基因组DNA中的基因是主要在基因表达的过程中，该mRNA是不稳定的，以构成信号。 RNA是非常迅速降解RNA酶酶终止信号。从生物体组织分离时，RNA是非常经常退化的表达之前在研究实验室研究。降解的RNA是不适合用于分析基因的表达。由于实验室工作人员不能参加手术，我们开发了一个过程，是很容易的，只需要外科医生恢复组织到合适的无RNA酶的解决方案。组织中的RNA中是稳定的，可以分析在该溶液中在室温下72小时后，没有任何降解的迹象。从标本中的RNA中纯化由一个标准化的方法涉及在氯化铯密度梯度超速离心分离后，被转移到实验室3。然后，RNA的质量，通过琼脂糖凝胶电泳，并通过RT-PCR评估的。 RNA纯化协议根据它们的密度是不同的DNA和RNA，并保证不被污染的RNA的DNA分子在基因表达研究中，将产生人造的信号的分离的分子的优点。此外，转移RNA（tRNA的），这是定量细胞内含量最丰富的RNA，分离到相同的物理属性从核糖体RNA（核糖体rRNA）和信使RNA（mRNA的），这两个最后一个是纯化过程中的最终产品。从制剂中的tRNA的去除是非常有用的，因为大多数的微阵列分析方案涉及使用反转录酶和RNA聚合酶而抑制tRNA的4-6。纯化的RNA的手术标本，使用标准协议的标记，杂交的微阵列芯片的结果进行了分析使用两个互补的方法，假发现率的方法，并使用了一种基于互信息与可视捷思上基于Web服务器Retinobase的7,8。

<table><tbody><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Aventi J-E</td><td></td></tr><tr><td>Rotor</td><td>Beckman Coulter</td><td>JS-5.3</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>LE 80K</td><td></td></tr><tr><td>Rotor</td><td>Beckman Coulter</td><td>SW41</td><td></td></tr><tr><td>Power supply</td><td>Biorad</td><td>Pac 3000</td><td></td></tr><tr><td>PolytronTM</td><td>Kinematica</td><td>PT 2100</td><td>Supplied with PT-DA 2105:2</td></tr><tr><td>Agarose gel electrophoresis device</td><td>Biorad</td><td>MiniGel Cell GT</td><td></td></tr><tr><td>Imaging System </td><td>Biorad</td><td>GelDoc-It Imager</td><td></td></tr><tr><td>5 ml sterile polyethylene tube</td><td>Greiner</td><td>115261</td><td></td></tr><tr><td>Sterile polyallomer centrifuge tube </td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Chemical reagents</td><td>Sigma</td><td></td><td>Molecular biology grade (RNase and DNase free products)</td></tr><tr><td>Cleaning Solution </td><td>VWR</td><td>RBS</td><td></td></tr><tr><td>Microarray chip</td><td>Affymetrix</td><td>Human U133 plus 2 array</td><td></td></tr></tbody></table>

transcriptomic analysis of human retinal surgical specimens using journal

视网膜脱离（RD）介绍视网膜色素上皮（RPE）的视网膜神经上皮层的分离。的视网膜色素上皮的光敏感的神经元，光感受器的正常功能是至关重要的。从视网膜色素上皮脱离，视网膜创建一个物理间隙充满了细胞外液。 RD启动细胞和分子的不良事件，严重影响视网膜神经上皮层和视网膜色素上皮，因为生理的离子和代谢产物的交换严重扰动。果为视野脱离自一个的快速reapposition的两种组织的查询结果恢复视力1的持续时间有关。 RD的治疗完全是手术。去除玻璃体凝胶（玻璃体切除术）之后是除去非必需的部分分离的区域，有利于视网膜脱离的视网膜周围。删除视网膜标本是无主财产 （无），因此通常被丢弃。要恢复RNA从这些手术标本，我们开发的程序允许RNA保护杂志在从手术块转移到实验室。而且，我们标准化的协议通过氯化铯超速离心纯化RNA，以确保适用于全局基因表达分析，纯化的RNA。通过RT-PCR和微阵列分析的RNA的质量进行了验证。对数据的分析示出了同时参与炎症和光感受器变性过程中RD。

作者:（ 图1）的程序允许我们从外科手术的块恢复视网膜标本纯化的RNA，并分析视网膜脱离的转录。视网膜脱离的查询结果中的单核细胞趋化CCL2，MCP1基因的上调和杆感光体转导基因GNAT1短波锥视蛋白OPN1SW和homeogene CRX（ 图2）的表达减少。 的CCL2的感应产生的炎症9，而同时减少，GNAT1，OPN1SW CRX是光感受器变性的结果，两个杆...

Watch this Scientific Journal Video about Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl at JoVE.com

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

我们使用了视网膜从retinectomy样本为转录组分析视网膜脱离。我们开发了一种程序，允许RNA保护手术块和实验室之间。我们的标准化的协议通过氯化铯超速离心法，以保证纯化的RNA是适合于微阵列分析中纯化RNA。

基因表达谱分析人视网膜手术标本杂志

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

transcriptomic-analysis-human-retinal-surgical-specimens-using

Research

JoVE Journal

Medicine

7.8K 次观看.  Institut National de la Santé et de la Recherche Médicale. 我们使用了视网膜从retinectomy样本为转录组分析视网膜脱离。我们开发了一种程序，允许RNA保护手术块和实验室之间。我们的标准化的协议通过氯化铯超速离心法，以保证纯化的RNA是适合于微阵列分析中纯化RNA。

视频: Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

人视网膜解剖与 RPE-脉络膜的蛋白质组分析

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

科研

生物化学

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

简化技术<em&gt;原位</em&gt;切除角膜和视网膜组织剜人眼地球

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

医学

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

技术处理眼睛的视网膜假体植入局部组织病理学分析

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

神经科学

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

生物学

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

基因表达谱分析人视网膜手术标本杂志

摘要

探索更多视频

此视频中的章节

人视网膜解剖与 RPE-脉络膜的蛋白质组分析

简化技术

技术处理眼睛的视网膜假体植入局部组织病理学分析

的vibratome切片小鼠视网膜准备感光文化

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

基因表达谱分析人视网膜手术标本杂志

摘要

探索更多视频

此视频中的章节

人视网膜解剖与 RPE-脉络膜的蛋白质组分析

简化技术

技术处理眼睛的视网膜假体植入局部组织病理学分析

的vibratome切片小鼠视网膜准备感光文化

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型