Name: transcriptomic analysis of human retinal surgical specimens using journal
Uploaded: 2013-08-14T13:00:00
Description: Watch this Scientific Journal Video about Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl at JoVE.com

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>...

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	<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>

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 border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <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?...

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  ...

Research

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Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...