Name: detection of micrornas in microglia by real-time pcr in normal cns and during neuroinflammation
Uploaded: 2012-07-23T13:00:00
Description: Watch this Scientific Journal Video about Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation at JoVE.com

这项工作是由美国国立卫生研究院以R01 NS071039-01A1研究资助支持。

1。从正常中枢神经系统小胶质细胞的分离小胶质细胞的分离以前修改3。 <ol><li>解剖和血流准备工具：剪刀，镊子，10毫升注射器27克针，15毫升Dounce匀浆（铁氟龙/玻璃），40微米尼龙细胞过滤器，40％和70％的Percoll方案，1×PBS。 </li><li>安乐死小鼠CO 2的相机快窒息（缺氧），并立即转移，手术操作和灌注的实验台。 </li><li>执行手术操作（用剪刀，镊子，70％?...

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	<li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Microglia+in+the+Healthy+Brain.">The Role of Microglia in the Healthy Brain.</a> J. Neurosci. 31, 16064-16069 (2011).</li><li>Graeber, M. B., Li, W., Rodriguez, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microglia+in+CNS+inflammation.">Role of microglia in CNS inflammation.</a> FEBS Lett. , (2011).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. 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N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546-3791 (2010).</li><li>Gabriely, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+21+promotes+glioma+invasion+by+targeting+matrix+metalloproteinase+regulators.">MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators.</a> Mol. 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Protoc. 1, 1947-1951 (2006).</li><li>Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia.">A simple magnetic separation method for high-yield isolation of pure primary microglia.</a> J. Neurosci. Methods. 194, 287-296 (2011).</li><li>Moltzahn, F., Hunkapiller, N., Mir, A. A., Imbar, T., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Throughput+MicroRNA+Profiling:+Optimized+Multiplex+qRT-PCR+at+Nanoliter+Scale+on+the+Fluidigm+Dynamic+ArrayTM+IFCs.">High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs.</a> J. Vis. Exp. (54), e2552 (2011).</li></ol>

最近显着的利益，考虑到小胶质细胞，这些细胞参与神经发炎和神经退行性疾病相关的许多病症。此外，在免疫系统和癌细胞分化的microRNA的作用显着增长在过去几年5,10。在非激活或休息的小胶质细胞的表型，在7正常的中枢神经系统的维护，我们曾报道了miR-124的作用，但其他类型的小分子RNA在小胶质细胞的表型和激活状态的作用仍然被发现。这将需要开发专门测量中的小胶质细?...

<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> 1×PBS </td><td> GIBCO公司</td><td> 14190 </td><td>可制取干元件实验室，pH值7.2-7.4 </td></tr><tr><td> 10X PBS </td><td> Thermo Scientific的</td><td> SH30258.02 </td><td>可制取干元件实验室，pH值7.2-7.4 </td></tr><tr><td> DMEM培养液，胎牛血清</td><td> GBCO </td><td> 11965，10438 </td><td></td></tr><tr><td> miRVana套件</td><td> Invitrogen公司</td><td> AM1561 </td><td></td></tr><tr><td> Percoll密度</td><td>西格玛</td><td> P4937-500毫升</td><td> 100毫升100％的Percoll准备通过混合10毫升10倍PBS和90毫升的Percoll自Sigma公司。 50毫升70％的Percoll准备35毫升100％的Percoll混合15毫升的DMEM 50毫升40％的Percoll准备通过混合20毫升100％的Percoll，30毫升的1X PBS </td></tr><tr><td> MOG 35-55肽</td><td> AnaSpec公司</td><td> 60130-5 </td><td></td></tr><tr><td>终审法院</td><td> DIFCO </td><td> 263810 </td><td></td></tr><tr><td>百日咳毒素</td><td>西格玛</td><td> P7208 </td><td></td></tr><tr><td> FCR封闭抗体</td><td> BD公司</td><td> 553142 </td><td>克隆2.4G2 </td></tr><tr><td>抗CD11b -体育单克隆抗体</td><td> BD公司</td><td> 553311 </td><td>可以用不同的荧光（如AF488） </td></tr><tr><td>抗CD45-APC-Cy7单克隆抗体</td><td> Biolegend </td><td> 103116 </td><td>可以用不同的荧光（如APC）的</td></tr><tr><td>多聚甲醛甲醛（16％） </td><td>环境管理体系公司</td><td> 15710 </td><td>稀释1×PBS 1:15 </td></tr><tr><td> 15％，简称TBE-尿素凝胶</td><td>生命科技公司</td><td> EC68855BOX </td><td></td></tr><tr><td>的TaqMan microRNA的反转录试剂盒</td><td>生命科技股份有限公司</td><td> 4366596 </td><td></td></tr><tr><td>的TaqMan通用PCR扩增预混</td><td>生命科技股份有限公司</td><td> 4304437 </td><td></td></tr><tr><td>的TaqMan microRNA的检测MMU的miR-124A </td><td>生命科技股份有限公司</td><td> 4427975编号001182 </td><td></td></tr><tr><td>的TaqMan microRNA的检测的snoRNA-55 </td><td>生命科技股份有限公司</td><td> 4427975编号001228 </td><td></td></tr><tr><td>无核酸酶水</td><td>生命科技股份有限公司</td><td> AM9937 </td><td>无核酸酶水</td></tr><tr><td> 384清晰的光学反应板</tD&gt; <td>生命科技公司</td><td> 4309849 </td><td>在不同的实时PCR仪器可以取代其他板块（如96孔板） </td></tr><tr><td> dounce均质（15毫升） </td><td>惠顿科学产品。 </td><td> 358044 </td><td>铁氟龙/玻璃</td></tr><tr><td> 40μ的尼龙细胞过滤</td><td>鹘</td><td> 352340 </td><td></td></tr><tr><td>大离心机</td><td> sorval </td><td>逆转录6000B </td><td>转子直径18.5厘米的</td></tr><tr><td>小离心机</td><td> Eppendorf公司</td><td> 5415ŕ </td><td>转子直径6.5厘米的</td></tr><tr><td>实时PCR仪</td><td>生命科技公司</td><td> AB 7900羟色胺</td><td>与其他仪器可以取代</td></tr><tr><td>流式细胞仪</td><td>屋宇署Biosciencess </td><td> LSR的二</td><td>与其他仪器可以取代</TD&gt; </tr><tr><td>流式细胞仪</td><td> BD公司</td><td> FACSAria </td><td>与其他仪器可以取代</td></tr><tr><td>的NanoDrop分光光度计</td><td> Thermo Scientific的</td><td>的NanoDrop 1000 </td><td></td></tr></tbody></table>

detection of micrornas in microglia by real-time pcr in normal cns and during neuroinflammation

小胶质细胞是驻留在中枢神经系统（CNS）的髓系细胞。这些细胞中发挥了重要作用，在许多疾病的病理与神经发炎，如多发性硬化症（MS）2。在一个正常的中枢神经系统胶质细胞表达巨噬细胞标记CD11b和休息型，如活化标志CD45的表达水平低的表现。在中枢神经系统的病理事件中，成为激活小胶质细胞所确定的上调CD45和其他标记3。在中枢神经系统的小胶质细胞的表型和功能的影响因素没有得到很好的研究。小分子RNA（miRNAs）是一个保守分子（〜22个核苷酸长），在许多正常的生理过程，如细 ​​胞生长和分化的4和5炎症等病症的越来越多的家庭。 miRNA的某些靶基因的表达下调的结合互补序列IR基因和发挥了重要作用，在激活先天免疫细胞，包括巨噬细胞和小胶质细胞7。为了调查定义的小胶质细胞的表型，生物功能，以区别于其他类型的巨噬细胞的小胶质细胞的miRNA介导的途径，它是重要的，特别是在居民中枢神经系统的胶质细胞不同亚群的microRNA表达定量评估。测量在中枢神经系统表达的miRNA的常用方法包括定量PCR技术，从整个神经组织和原位杂交。然而，从整个组织匀浆PCR定量不允许在小胶质细胞的miRNA表达的评估，这代表只有5-15％的神经组织的细胞。 原位杂交允许评估组织切片中的特定类型的细胞的microRNA表达，但这种方法是不完全定量。在这份报告中，我们描述了定量和SENSitive实时PCR检测中的小胶质细胞在正常的中枢神经系统或神经发炎使用实验性自身免疫性脑脊髓炎（EAE）小鼠模型，为MS分离miRNA的方法。所描述的方法将是有益的，在正常的中枢神经系统，或在与各种病症，包括MS，中风，跌打损伤，阿尔茨海默氏症和脑肿瘤相关的神经发炎的小胶质细胞的microRNA表达水平来衡量的。

Watch this Scientific Journal Video about Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation at JoVE.com 

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation (Video) | JoVE 

小胶质细胞的居住提供第一线的防御和免疫监视中枢神经系统的巨噬细胞。 microRNA是调节分子，包括巨噬细胞的激活和分化的许多生理过程中发挥了重要作用。在这篇文章中，我们描述了在小胶质细胞的小分子RNA的测量方法。

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Research

JoVE Journal

Immunology and Infection

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

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在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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Culturing Microglia from the Neonatal and Adult Central Nervous System

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在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

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