Name: obtaining high quality rna from single cell populations in human postmortem brain tissue
Uploaded: 2009-08-06T19:00:00
Duration: 18 min 17 s
Description: Watch this Scientific Journal Video about Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue at JoVE.com

<p class="jove_content">我们非常感谢他们的研究中使用的试剂的慷慨捐赠分子器件分析技术/大角星。我们也感谢哈佛组织资源中心提供死后的人脑<strong>组织的资金</strong>来源：RO1MH76060和P50MH080272。 </p>

<ol>
	<li>Imbeaud, S., Graudens, E., Boulanger, V., Barlet, X., Zaborski, P., Eveno, E., Mueleer, O., Schroeder, A., Auffray, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> <em style='font-style: italic'>Nucleic Acids Res</em>. <b>33</b>, e56-e56 (2005).</li></ol>

<p class="jove_content">生产这种视频文章是由生命科技赞助。 </p>

<p class="jove_content">在这里，我们尝试自定义协议的异构死后脑组织使用的大角星XT系统和相关的RNA提取试剂盒提取同质的细胞群。如上所述，我们已作了一些修改，由该公司提供的的原始协议，以最大限度地提高RNA的完整性。成功捕获单细胞完全取决于优化的步骤之前，捕捉，以达到最大细胞具有良好的RNA。这些步骤包括：与组织切片及染色相关的各种参数。总之，这些方面组织编制包括：1）降低温度梯度时切片，2）放置2节每张幻灯片，3）在冰上表演的所有脱水步骤，4）加入RNase抑制剂，污渍，5）在最后100％的乙醇至3分钟的脱水时间。 </p><p class="jove_content">方面捕捉，我们认为结合CapSure HS帽与宏设置软件，应确保达到最佳的效果。湿度控制的环境中捕捉单个细胞的关键，在高湿度的大幅度降低组织的“电梯”。高温也可以对RNA质量的负面影响。 </p><p class="jove_content">在与PicoPure套件的RNA分离协议中，有两个洗步骤。重要的是要检查所有的洗涤缓冲液，然后转移到另一微量RNA的洗脱管中删除，您的最终样品中存在的缓冲区的任何将导致开始在更短的RNA扩增。为了确保这一点，我们离心机为2.5分钟，而不是在所提供的协议建议的2分钟的最后一次洗涤步骤。 </p><p class="jove_content">在大多数情况下，线性放大协议是根据制造商的指示。但是，有两个关键点，应考虑使用此协议时：1）尽量限制通过样品的过剩转移RNA的损失之间的管，只使用0.5毫升管结​​合提供了一个在热循环仪0.5毫升块; 2）当样品转移到纯化柱纯化的cDNA，请务必降速后的第一个转移的样品管。这将导致额外的1 - 2ul样品添加到纯化柱，因此可以增加的cDNA恢复。 </p><p class="jove_content">当自己的有限ARNA样本的生物素标签的Turbo结束标记试剂盒的使用，我们建议增加一个额外的几微升的水的离心步骤之前，增加标记的RNA产量列中提取。一个额外的离心分钟，也将有助于在这方面，特别是如果你与退化的组织，如FFPE组织工作。 </p><p class="jove_content">我们有信心，这个协议将使用户隔离单异质死后脑组织内的细胞群，如小的均质结构，。这样可以减少周围结构，位于大脑内的各层的其他细胞类型的稀释效应，导致对正在调查中特定的细胞类型有针对性的结果。由于所产生的RNA的质量是好的芯片研究不够，我们就可以开始针对特定的分子特征，特定的细胞在疾病状态影响的人口。 </p>

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>4.5&#x201D; Forceps</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-392</td>
<td>&nbsp;</td>
<tr>
<td>Arcturus XT&trade; Laser-Capture Microdissection (LCM) System</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>13821-00</td>
<td>&nbsp;</td>
<tr>
<td>CapSure&trade; HS LCM Caps</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0214</td>
<td>&nbsp;</td>
<tr>
<td>CapSure&reg; Macro LCM Caps</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0211</td>
<td>&nbsp;</td>
<tr>
<td>Experion&trade; Automated Electrophoresis Station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7001</td>
<td>&nbsp;</td>
<tr>
<td>Experion&trade; RNA HighSens Chips</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7155</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&#x2122; RNA HighSens Reagents</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7156</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&#x2122; RNA StdSens Chips</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7153</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&trade; RNA StdSens Reagents</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7154</td>
<td>10 chips</td>
</tr>
<tr>
<td>Falcon&reg;  polypropylene Conical tube</td>
<td>&nbsp;</td>
<td>Becton-Dickinson Labware</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>GeneAmp&reg; thin-walled reaction tube with domed cap</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8010611</td>
<td>&nbsp;</td>
<tr>
<td>GeneChip&reg; Human X3P array</td>
<td>&nbsp;</td>
<td>Affymetrix</td>
<td>900516</td>
<td>6 chips</td>
</tr>
<tr>
<td>Histogene&reg; LCM Frozen Section Staining kit</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0401</td>
<td>For staining solution, jars, ethanols and Xylene</td>
</tr>
<tr>
<td>Histogene&trade; LCM slides</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>12231-00</td>
<td>&nbsp;</td>
<tr>
<td>Micro Slide Box</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48444-004</td>
<td>&nbsp;</td>
<tr>
<td>Microm HM 505E cryostat</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22-050-350</td>
<td>Catalogue number for similar product as current product no longer available</td>
</tr>
<tr>
<td>Microprocessor Controlled 280 series Water bath</td>
<td>&nbsp;</td>
<td>Thermo Electron Corporation</td>
<td>51221048</td>
<td>Model 282</td>
</tr>
<tr>
<td>Molecular Sieve</td>
<td>&nbsp;</td>
<td>EMD&trade;</td>
<td>MX1583L-1</td>
<td>&nbsp;</td>
<tr>
<td>NanoDrop 1000 Spectrophotometer</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>n/a</td>
<td>The NanoDrop 1000 is not available anymore, but the NanoDrop 2000 is comparable.</td>
</tr>
<tr>
<td>Nuclease-free water</td>
<td>&nbsp;</td>
<td>Ambion&reg;</td>
<td>AM9932</td>
<td>&nbsp;</td>
<tr>
<td>PEN Membrane glass slides</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0522</td>
<td>&nbsp;</td>
<tr>
<td>PicoPure&reg; RNA Isolation kit</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0204</td>
<td>&nbsp;</td>
<tr>
<td>RiboAmp&reg;HS<sup>PLUS</sup> with Biotin labeling</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0511B</td>
<td>12 reactions Includes TURBO biotin labeling&trade; kit</td>
</tr>
<tr>
<td>RNase Zap&reg;</td>
<td>&nbsp;</td>
<td>Ambion&reg;</td>
<td>AM9780/2</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase Set</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>79254</td>
<td>&nbsp;</td>
<tr>
<td>RNasin&reg; Plus</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N261B</td>
<td>&nbsp;</td>
<tr>
<td>SuperScript&trade; III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18080-044</td>
<td>&nbsp;</td>		</tbody>
		</table>
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obtaining high quality rna from single cell populations in human postmortem brain tissue

<p class="jove_content">我们提出要调查见于精神分裂症患者颞上回在灰质减少，通过询问在第三层锥体神经元的基因表达图谱。这是众所周知的，大脑皮层异常庞杂的结构，包括不同地区，层次和类型的细胞，每一个特点是由不同的细胞和分子组成，因此，基因差异表达谱。为了规避组织异质性的混杂的影响，我们用激光捕获显微切割（LCM），以隔离特定的细胞类型，即锥体神经元。 </p><p class="jove_content">使用大角XT的LCM系统Histogene染色液染色约500锥体神经元被抓获。 RNA，然后从捕获的细胞中分离出，并经历了两轮基于T7使用大角/套分子器件的线性放大。的Experion LabChip（BIO - RAD）凝胶电泳表明，良好的质量（M）RNA，成绩单扩展微阵列所需的过去600nt的长度。获得表达量平均51μg可接受的平均样品纯度，A260/280比值为2.5，表示。异形使用基因芯片由Affymetrix公司的人力X3P探针阵列基因表达。 </p>

Watch this Scientific Journal Video about Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue at JoVE.com

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

<p class="jove_content">我们描述一个过程，利用激光捕获显微切割分离和提取RNA从一个同质的细胞群，锥体神经元，在上级死后人类大脑颞上回第三层。其后，我们线性放大（T7基于）的mRNA，Affymetrix公司人类X3P芯片杂交样品。</p>

人死后脑组织中的单细胞群获得高品质的RNA

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