Name: rna-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells
Uploaded: 2013-02-13T13:00:00
Description: Watch this Scientific Journal Video about RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells at JoVE.com

作者会喜欢到感谢博士斯蒂芬金斯莫尔和的儿科基因组医学中心儿童慈悲医院和诊所为他们的计算集群为我们的数据分析的，Illumina公司的现场服务团队（伊丽莎白·博耶，斯科特·库克和马克库克）和技术顾问团队，为他们的快速反应和下一代DNA测序仪，HiScanSQ，和数据质量分析的运行有用的建议。这项工作是支持的，部分由美国国立卫生格兰特HL080042（SQY）启动基金和养老的儿童慈善医院和诊所在堪萨斯城，密苏里大学（SQY）。

<html>概述本协议的流程图显示在图1。 1。处理过的细胞凝血酶，总RNA的提取，质量评价和定量RNA <ol><li>培养人肺微血管内皮细胞（HMVEC-LBL）90-100％汇合在EGM-2培养基的6孔板中，5％FBS，生长因子和抗生素（龙沙，猫＃CC-3202）。 </li><li>变更媒体饥饿媒体（0％FBS）30分钟前，用凝血酶处理。 </li><li>治疗的细胞用0.05 U / ml凝血酶或留下未处理的作为控制6小时，在37℃和5...

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	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat. Rev. Genet. 10, 57-63 (2009).</li><li>Shendure, J., Ji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+DNA+sequencing.">Next-generation DNA sequencing.</a> Nat. Biotechnol. 26, 1135-1145 (2008).</li><li>Marioni, J. C., Mason, C. E., Mane, S. 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关键步骤 RNA处理核糖核酸酶会降低，即使是最优质的RNA，因此，护理时必须采取相应的隔离，储存和使用的RNA 10。总是戴手套，以防止污染由核糖核酸酶中发现人的手。手套应经常更换，尤其是接触皮肤后，门把手或其他常见的表面。一组吸液管应专门的RNA工作，所有的技巧和管应无RNA酶。 RNA分离和下游的应用程序，应进行低流量区域，常规的RNase-扎普如...

<table border="1"><tbody><tr><td width="183"> 试剂和设备 </td><td width="85"> 公司 </td><td width="185"> 目录号 </td><td width="359"> 评论 </td></tr><tr><td>人肺微血管内皮细胞</td><td>龙沙</td><td> CC-2815 </td><td></td></tr><tr><td>龙沙，子弹套件</td><td>龙沙</td><td> CC-3202 </td><td>包含EGM-2，FBS，生长因子和抗生素</td></tr><tr><td>凝血酶</td><td>西格玛</td><td> T4393 </td><td></td></tr><tr><td> Ambion公司MIR瓦纳套件</td><td> Life Technologies公司</td><td> AM 1560 </td><td></td></tr><tr><td>核糖核酸酶-ZAP </td><td> Life Technologies公司</td><td> AM9782 </td><td></td></tr><tr><td> Experion的StdSens RNA </td><td> Bio-Rad公司</td><td> 700-7103 </td><td></td></tr><tr><td> TruSeq RNA的制备试剂盒</td><td> Illumina公司</td><td> FC-122-1001 </td><td></td></tr><tr><td> AMPureXP珠</td><td> Beckman Coulter公司</td><td> A63881 </td><td></td></tr><tr><td>标逆转录酶II </td><td> Life Technologies公司</td><td> 18064-014 </td><td></td></tr><tr><td> Experion的DNA 1K </td><td> Bio-Rad公司</td><td> 700-7107 </td><td></td></tr><tr><td> QuantiTect SyberGreen </td><td> Qiagen公司</td><td> 204163 </td><td></td></tr><tr><td> PE集群生成工具包</td><td> Illumina公司</td><td> PE-401-3001 </td><td></td></tr><tr><td> PhiX控制工具包</td><td> Illumina公司</td><td> FC110-301 </td><td></td></tr><tr><td> 200周期SBS套件</td><td> Illumina公司</td><td> FC-401-3001 </td><td></td></tr><tr><td> HiScanSQ * </td><td> Illumina公司</td><td> SY-103-2001 </td><td></td></tr><tr><td>芝加哥期货交易所</td><td> Illumina公司</td><td> SY-301-2002 </td><td></td></tr><tr><td>定量PCR机 - Viia7台</td><td> Life Technologies公司</td><td>型号＃VIIA7 /设备＃10631261 </td><td>或同等学历</td></tr><tr><td> Experion系统</td><td> BIO RAD </td><td> 7007001 </td><td>生物分析仪是一种替代系统</td></tr><tr><td>分光光度计</td><td> Bio-Tek的</td><td>大纪元微孔板分光光度计</td><td>或同等学历</td></tr><tr><td>离心机 - SORVALL传奇XTR </td><td> Thermo Scientific的</td><td> 75004521 </td><td>或同等学历</td></tr><tr><td>磁性底座</td><td> Life Technologies公司</td><td> AM10027 </td><td></td></tr><tr><td> 96孔热循环仪</td><td>一般实验室供应商</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 表3列出主要试剂和主要电子quipment。 *，在视频中，HiSeq1000证明而不是HiScanSQ的。 </td></tr></tbody></table>

rna-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells

通过测量细胞中的mRNA水平的基因表达的特征是确定如何转录机制的细 ​​胞受到外部信号（ 如药物治疗），或不同的细胞如何健康状态和疾病状态之间的一个有用的工具。随着新一代DNA测序技术的出现和不断改进，RNA测序（RNA-seq的）已成为越来越受欢迎的转录组分析的方法进行编目所有种类的成绩单，以确定所有基因表达的转录结构和量化的表达水平的变化在一个给定的细胞，组织或生物体1,2的总组转录。 RNA-Seq是逐渐取代DNA微阵列转录组分析的首选方法，因为它有一个完整的转录组分析的优势，提供一个数字型数据（拷贝数的任何誊本），而不是依靠任何已知的基因组sequenCE 3。 在这里，我们提出了一个完整的，详细的协议，适用个人资料转录RNA-seq的人肺微血管内皮细胞或无凝血酶治疗。该协议是基于我们最近发表的研究报告，题为“RNA-seq的揭示新型转录的基因及其亚型在人肺微血管内皮细胞凝血酶治疗，”4中，我们成功地完成了第一个完整的人肺微血管内皮细胞的转录组分析与凝血酶使用RNA-seq的治疗。进一步的实验，取得了前所未有的资源，获得洞察的分子机制，凝血酶介导的内皮功能紊乱的炎症性疾病，癌症，糖尿病和冠状动脉心脏疾病的发病机制，并为这些疾病的治疗目标，以提供潜在的新的线索。 此协议的描述性文本被分成四个部分。第一部分介绍了人肺微血管内皮细胞的凝血酶和RNA的提取，质量分析和定量分析的治疗。第二部分介绍了图书馆的建设和测序。第三部分描述了数据分析。第四部分描述的RT-PCR验证试验。显示了有代表性的几个关键步骤的结果。在讨论中提供有用的提示或预防措施，以提高成功的关键步骤。虽然该协议使用人肺微血管内皮细胞用凝血酶，它可以是广义在哺乳动物和非哺乳动物的细胞和组织中具有不同刺激物或抑制剂，或在细胞或组织之间的健康状态来比较转录处理的档案中转录和疾病状态。

对于步骤1：28秒：18比传统使用的RNA的降解是一个指标。在理想的情况下，28秒峰值应具有约两倍的面积的18带（2）的比率，但是这理想比例往往不能在实践中看到。此外，28秒：18秒从分光光度测定方法得到的比率可以低估的量的RNA的降解。为了更精确地量化的退化，并因此的RNA样品的质量，的Experion系统计算的RNA质量指示符（RQI）号码。 RQI算法RNA样品的电泳图谱进行比较，从一系?...

Watch this Scientific Journal Video about RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells at JoVE.com

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

该协议提供了一个完整而详细的程序，申请一个功能强大的下一代DNA测序技术，RNA-seq技术，在人肺微血管内皮细胞或无凝血酶治疗的个人资料转录。这个协议是一般化到各种细胞或组织中的影响不同的试剂或疾病状态。

RNA-seq的凝血酶处理和控制人肺微血管内皮细胞的转录组分析

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

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