This work is supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC: 418391-2012). AC receives a scholarship from the Centre th&#233;matique de recherche en Neurosciences (CTRN). HDB is funded by a scholarship from the Fonds de Recherche en Sant&#233; du Qu&#233;bec (FRSQ) and ML is a FRSQ Chercheur-Boursier.

注:该实验是按照加拿大指南实验动物的护理和使用进行，并经拉瓦尔大学动物保护委员会。 1.样品制备注意:在这个例子中，我们使用鼠标大脑在出生后第1天。 <ol><li>使用低温麻醉在碎冰为3至4分钟为幼仔，然后在冰冷的L15培养基中尽可能快地解剖大脑。执行在2分钟内该步骤。 </li><li>在嵌入模具（物资比照列表）转让解剖大脑和填充照顾以定向标本所需的位置冰冻组织包埋媒体。在-80℃下在液态氮和存储立即冻结标本。执行在30秒内这一步。 注:样品可以在不损害RNA的质量被储存几个月。 </li></ol> 2.切片<ol><li>治疗膜COA泰德玻片（ 比照材料清单）与RNA酶表面净化解决方案，以消除RNA酶的任何踪迹。在DEPC水冲洗的幻灯片，让他们干。可替代地，烘烤幻灯片过夜，在200℃。 </li><li>在转让之前与表面去污RNA酶溶液清洗低温恒温器冷冻样品。设置低温恒温室的温度在-20℃和切片标本在10微米厚。收集组织切片上的膜涂层的幻灯片，让他们干10分钟染色前。可替代地，存储幻灯片在-80℃直至染色。 </li></ol> 3.准备染色解决方案<ol><li>准备所有解决方案只是在实验开始前，并使用在所有染色溶液RNA酶抑制剂（除洗涤溶液），以防止RNA降解。 </li><li>制备缓冲溶液。使用在所有的解决方案同样的缓冲液（无RNA酶的磷酸盐缓冲盐水（PBS组成），补充1％的BSA，0.2％的Triton和2％RNA酶抑制剂）。对于每个幻灯片，准备400微升缓冲液（200微升用于初级和200微升的二次抗体）。 </li><li>制备固定剂溶液:用70％的乙醇保持在-20℃。保持组织固定过程最少，以避免急剧减少，在RNA提取收率。 注意:可替换地，可使用补充的5％乙酸的95％的乙醇溶液保持在-20℃。 </li><li>通过在缓冲溶液稀释的第一抗体制备第一抗体溶液。为多巴胺能神经元的标记，可使用靶向酪氨酸羟化酶（TH，速率限制酶对多巴胺生产）的抗体。使用高浓度的抗体，以补偿快速孵育时间。使用TH抗体在稀释1:25。 注:在正常免疫协议，该抗体给出的稀释度1孵育过夜，当强信号:1000。在这个协议中，它是用来40X更concentrat由于短的孵育时间编由于高浓度的抗体用于此方法中，为了检查非特异性标记的任何增加执行标准的免疫染色并联。 </li><li>通过在缓冲溶液稀释的二级抗体制备二级抗体溶液。使用生物素化的第二抗体以1:1的浓度:100。 注意:在这个例子中，我们使用抗兔生物素化的抗体。 </li><li>制备的抗生物素蛋白 - 生物素复合物（ABC）对生物素化的二级抗体的检测溶液。使ABC溶液通过将化合物A和化合物B以1:1的浓度:100稀释于DEPC PBS中补充有2％的RNA酶抑制剂。将其添加到幻灯片之前制备抗生物素蛋白 - 生物素复合物30分钟。 </li></ol> 4.染色<ol><li>通过快速轻弹它们在空气中解冻，在从-80时℃的一个或两个幻灯片（或使用鼓风机）构成。用疏水性的环绕部分屏障笔，让干燥的几秒钟。 <ol><li>把载玻片在冷的固定剂溶液不超过5分钟，在-20℃下。摇一次在空气中，以除去过量的固定剂溶液然后，迅速浸载玻片于DEPC PBS 6-8次，洗涤。 </li><li>轻弹幻灯片以去除多余的DEPC PBS的。确保幻灯片除霜步骤不超过5分钟。 </li></ol></li><li>在一个干净的托盘处的幻灯片（预处理用表面RNA酶去污溶液，以消除RNA酶的任何迹线），并把200μl的初级抗体溶液的每个滑板（兔抗TH）上，在室温下10分钟。浸迅速滑动于DEPC PBS中3次，每次洗涤和轻弹载玻片一次以除去过量的DEPC PBS中。 </li><li>把200μl的第二抗体溶液的每张幻灯片上为6分钟，在室温下进行。浸迅速滑动3倍于DEPC PBS中洗涤并轻弹载玻片以除去过量的DEPC PBS中。 </li><li>把200微升的ABC的SOLU灰每张幻灯片在室温下4分钟上。浸迅速滑动于DEPC PBS中3次，每次洗涤和轻弹载玻片以除去过量的DEPC PBS中。 </li><li>准备DAB（3,3&#39;二氨基联苯胺）培养的ABC解的4分钟内解决。使用在试剂盒（DAB过氧化物酶底物试剂盒）提供的试剂。拌1滴提供缓冲液中，1滴DAB溶液和1滴30％的H 2 O 2中的DEPC水2.5毫升。通过反转混合溶液。 </li><li>取196微升的DAB解决方案，只是在幻灯片上蔓延之前添加的RNA酶抑制剂的解决方案4微升（2％）。在一个干净的托盘（预处理表面RNA酶净化解决方案）的地方幻灯片，把200微升的DAB解决方案的幻灯片。 注:DAB的解决方案应该反应within1或2分钟。 </li><li>浸迅速滑动于DEPC PBS中3次，每次洗涤和放幻灯片几秒钟的无水乙醇。通过轻弹它们在空气干燥的幻灯片（或使用鼓风机）。&lt;BR /&gt;注:幻灯片可以储存在-80°C，以备将来使用或处理的LCM。 </li></ol> 5.激光捕获显微切割<ol><li>通过轻弹它们在空气中快速地解冻幻灯片（或使用鼓风机）构成。确保幻灯片除霜步骤不超过5分钟。 </li><li>进到LCM。放置幻灯片与面向收集管帽的样本面，在显微镜下。选择适合样品的最佳放大倍率。调节焦距以查看感兴趣的细胞。 <ol><li>限定在切割区域，并开始用激光切割。收集解剖的细胞或组织片在收集管帽填充有50微升裂解缓冲液（从RNA提取试剂盒）。确保LCM的步骤不超过30分钟。 </li></ol></li><li>提取使用RNA提取试剂盒（ 比照物料清单）分离细胞的总RNA。按照制造商的协议。 <ol><li>在42℃孵育收集的细胞进行30分钟至裂解细胞。先决条件，加入250微升调理缓冲器的RNA纯化柱。负载50微升乙醇溶解的细胞。加载100微升裂解的细胞到一个预条件纯化塔。 </li><li>离心柱在16000×g离心，在室温下2分钟。用水洗两次缓冲洗柱。洗脱在无RNA酶的水的最小推荐量（11微升）（未DEPC处理，因为它可能与生物分析器干扰）。保持2微升到测试质量。 注:所有的解决方案和试剂的试剂盒中提供。 </li></ol></li></ol> 6. cDNA合成和定量RT-PCR <ol><li>反向转录使用逆转录酶和寡（dT）的RNA样品从激光捕获多巴胺能细胞分化成互补DNA。按照制造商的协议（ 比照材料清单）。 </li><li>使用1微升cDNA的定量RT-PCR扩增与基因特异性引物。建立PCR意图在20μl体积与单组分热启动反应混合物用于定量PCR ctions如制造商推荐的。进行一式三份的每个反应中。 <ol><li>对于这里描述的实验中，使用以下引物对:GAPDH-F（5&#39;-CCA CCC AGA AGA CTG TGG的AT-3&#39;）/ GAPDH-R（5&#39;-GGA TGC AGG GAT GAT GTT CT-3&#39;）; RPL13A-F（5&#39;-ACA GCC ACT CTG GAG GAG AA-3&#39;）/ RPL13A-R（5&#39;-CTG C​​CT GTT TCC GTA ACC TC-3&#39;）; TH-F（5&#39;-CAG TGG AGG ATG TGT CTC-3&#39;）/钍-R（5&#39;-GAA AAT CAC GGG CAG ACA G-3&#39;）; AADC-F（5&#39;-CAT GAG AGC TTC TGC CCT TC -3&#39;）/ AADC-R（5&#39;-GGA TGT GGT CCC CAG TGT AG -3&#39;）。 </li></ol></li><li>使用以下的快速循环参数进行定量RT-PCR扩增:95℃下5分钟，随后10秒的45个循环95℃，10秒，在60℃，10秒72℃。使用仪器的默认设置determi进行熔解曲线分析NE同质产品的形成。 </li><li>分析使用内置的软件中的数据。 <ol><li>正常化的DA标记基因的Th和AADC的表情值与这两个参考基因GAPDH和RPL13A的表达，以获得相对表达水平如前面14所描述。 </li></ol></li></ol>

<ol>
	<li>Chung, C. Y., 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+transcription+factor+orthodenticle+homeobox+2+influences+axonal+projections+and+vulnerability+of+midbrain+dopaminergic+neurons.">The transcription factor orthodenticle homeobox 2 influences axonal projections and vulnerability of midbrain dopaminergic neurons.</a> Brain : a journal of neurology. 133 (Pt 7), 2022-2031 (2010).</li><li>Cheng, L., 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=Laser-assisted+microdissection+in+translational+research:+theory,+technical+considerations,+and+future+applications.">Laser-assisted microdissection in translational research: theory, technical considerations, and future applications.</a> Applied immunohistochemistry &amp; molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry. 21 (1), 31-47 (2013).</li><li>Decarlo, K., Emley, A., Dadzie, O. E., Mahalingam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection:+methods+and+applications.">Laser capture microdissection: methods and applications.</a> Methods in molecular biology. 755, 1-15 (2011).</li><li>Espina, V., Heiby, M., Pierobon, M., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+technology.">Laser capture microdissection technology.</a> Expert review of molecular diagnostics. 7 (5), 647-657 (2007).</li><li>Fend, F., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+pathology.">Laser capture microdissection in pathology.</a> Journal of clinical pathology. 53 (9), 666-672 (2000).</li><li>Chadi, G., Maximino, J. R., de Oliveira, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+molecular+histology+to+study+glial+influence+on+neurodegenerative+disorders.+Focus+on+recent+developed+single+cell+laser+microdissection.">The importance of molecular histology to study glial influence on neurodegenerative disorders. Focus on recent developed single cell laser microdissection.</a> Journal of molecular histology. 40 (4), 241-250 (2009).</li><li>Liu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+the+tissue+biorepository.">Laser capture microdissection in the tissue biorepository.</a> Journal of biomolecular techniques : JBT. 21 (3), 120-125 (2010).</li><li>Baskin, D. G., Bastian, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-laser+capture+microdissection+of+rat+brain+neurons+for+real+time+quantitative+PCR.">Immuno-laser capture microdissection of rat brain neurons for real time quantitative PCR.</a> Methods in molecular biology. 588, 219-230 (2010).</li><li>Brown, A. L., Smith, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+RNA+preservation+for+immunolabeling+and+laser+microdissection.">Improved RNA preservation for immunolabeling and laser microdissection.</a> RNA. 15 (12), 2364-2374 (2009).</li><li>Brown, A. L., Brown, A. L., Day, T. A., Dayas, C. V., Smith, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purity+and+enrichment+of+laser-microdissected+midbrain+dopamine+neurons.">Purity and enrichment of laser-microdissected midbrain dopamine neurons.</a> BioMed research international. 2013, 747938 (2013).</li><li>Murakami, H., Liotta, L., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IF-LCM:+laser+capture+microdissection+of+immunofluorescently+defined+cells+for+mRNA+analysis+rapid+communication.">IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication.</a> Kidney international. 58 (3), 1346-1353 (2000).</li><li>Greene, J. G., Dingledine, R., Greenamyre, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+rat+midbrain+dopamine+neurons:+implications+for+selective+vulnerability+in+parkinsonism.">Gene expression profiling of rat midbrain dopamine neurons: implications for selective vulnerability in parkinsonism.</a> Neurobiology of disease. 18 (1), 19-31 (2005).</li><li>Chung, C. Y., Koprich, J. B., Endo, S., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Endogenous+Serine/Threonine+Protein+Phosphatase+Inhibitor,+G-Substrate,+Reduces+Vulnerability+in+Models+of+Parkinson's+Disease.">An Endogenous Serine/Threonine Protein Phosphatase Inhibitor, G-Substrate, Reduces Vulnerability in Models of Parkinson's Disease.</a> Journal of Neuroscience. 27 (31), 8314-8323 (2007).</li><li>Pfaffl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Quantification+strategies+in+real-time.">Chapter 3, Quantification strategies in real-time.</a> AZ of quantitative PCR. , (2004).</li><li>Smolinski, D., Blessenohl, M., Neubauer, C., Kalies, K., Gebert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+novel+ultra-short+immunolabeling+method+for+high-quality+mRNA+preservation+in+laser+microdissection+and+real-time+reverse+transcriptase-polymerase+chain+reaction).">Validation of a novel ultra-short immunolabeling method for high-quality mRNA preservation in laser microdissection and real-time reverse transcriptase-polymerase chain reaction).</a> The Journal of MolecularDiagnostics. 8 (2), 246-253 (2006).</li><li>Podgorny&#301;, O. V., Lazarev, V. N., Govorun, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+for+biology+and+medicine.">Laser microdissection for biology and medicine.</a> Tsitologiia. 54 (5), 381-389 (2012).</li><li>Vandewoestyne, M., ewoestyne &amp; Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+forensic+research:+a+review.">Laser capture microdissection in forensic research: a review.</a> International journal of legal medicine. 124 (6), 513-521 (2010).</li><li>Shapiro, J. P., 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+quantitative+proteomic+workflow+for+characterization+of+frozen+clinical+biopsies:+laser+capture+microdissection+coupled+with+label-free+mass+spectrometry.">A quantitative proteomic workflow for characterization of frozen clinical biopsies: laser capture microdissection coupled with label-free mass spectrometry.</a> Journal of. 77, 433-4340 (2012).</li><li>Domazet, B., Maclennan, G. T., Lopez-Beltran, A., Montironi, R., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+the+genomic+and+proteomic+era:+targeting+the+genetic+basis+of+cancer.">Laser capture microdissection in the genomic and proteomic era: targeting the genetic basis of cancer.</a> International journal of clinical and experimental pathology. 1 (6), 475-488 (2008).</li></ol>

作者什么都没有透露。

这种方法的最关键的一点在于，同时防止RNA降解标记所关注的细胞。如RNA酶是活性的水溶液，减少温育时间提高的RNA保护11,15。所用的所有解决方案必须用DEPC灭活RNA酶处理。所有的材料和表面需要与表面RNA酶去污溶液，以防止RNA酶污染处理。最后，在这些条件下，在所有的溶液中加入RNA酶抑制剂是有效的，从而降低维护的RNA。在使用过程中抗体孵育施加高盐条件先前已经证明是有效的在?...

大脑是由大量不同形成的复杂网络的不同神经元类型。这些神经元根据其形态，连接性和基因表达模式1组织在不同的组和子组。微阵列的发展，新一代测序，和定量RT-PCR技术提供给在不同的生物的上下文1,2-比较神经元群体的基因表达图谱的可能性。这些敏感分析需要的利益，同时保持RNA的完整性2-4细胞类型的精确鉴定和分离。荧光激活细胞分选（FACS）技术已被广泛地用于分离根据细胞表面标志物和/或形态的特定细胞类型。流式细胞仪需要一个细胞解离步骤之前进行排序，这导致空间分辨率5的完全丢失。许多神经元的亚群是彼此可分辨根据它们在第解剖学分布Ë大脑。施加薄的脑切片的激光捕获显微切割提供了用于细胞以高空间分辨率6-8的特定隔离的适当选项。 LCM的一个主要限制是需要确定基于形态学标准或荧光蛋白记者基因工程在动物模型中所关注的细胞。新技术的发展，以执行保存RNA的完整性，与LCM结合快速免疫标记方法，现在允许细胞亚群的隔离进行基因分析实验。 这一方法已被预先执行，并且在几个出版物1,9-13简单说明。在这里，我们证明了详细的程序通过组合快速免疫标记与LCM获得高品质的RNA的细胞的一个复杂的组织结构的特定子集。我们将展示如何执行关键的关键步骤，以获得最大的RNA恢复，避免RNA降解，因为它可能标志贴着地影响基因表达分析。 对于该协议的示范，从一个天大的小鼠中脑多巴胺能神经元的目标。多巴胺能神经元可以使用针对酪氨酸羟化酶（TH）抗体，对多巴胺合成的限速酶免疫标记。以下TH免疫染色，从个人或多巴胺能神经元的组然后可以使用LCM隔离。显微细胞被收集在裂解缓冲液，和RNA是使用RNA分离试剂盒提取。质量和提取RNA的量使用的是生物分析仪测量5。随后可以进行定量RT-PCR检测RNA测序，微阵列，或2,4,6-:使用基因表达的进一步分析。作为一个例子，一个两步QRT-PCR证明在激光捕获分离多巴胺能域。两个多巴胺能神经元标记基因的表达水平相对定量说明了这一协议的选择性。

<table border="0" cellpadding="0" cellspacing="0" width="895">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td style="width:420px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Membrane coated slides&#160;</td>
			<td style="width:148px;">Leica Biosystems</td>
			<td style="width:99px;">11505158</td>
			<td>MembraneSlides PEN-Membrane 2,0 &#181;m; 50 pcs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Surface RNAse decontamination solution</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">AM9780</td>
			<td>RNaseZap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Fixative</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td>70% Ethanol kept at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Anti-TH&#160;</td>
			<td style="width:148px;">Pel-Freez</td>
			<td style="width:99px;">P40101</td>
			<td>&#160;1:25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNAse free buffer</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td>DEPC PBS + 1% BSA+0.02% triton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNAse inhibitor</td>
			<td style="width:148px;">Promega</td>
			<td style="width:99px;">N2615</td>
			<td>Rnasine Plus Rnase Inhibitor 40 kU/ml (stock)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Anti-rabbit biotinylated</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">BA-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Vectastain Elite ABC kit Standard</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">PK-6100</td>
			<td>8&#181;l/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">DAB Peroxidase Substrate Kit&#160;</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">SK-4100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNA isolation kit</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">KIT0204</td>
			<td>Arcturus PicoPure RNA Isolation Kit</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">H2O2 30%</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">HC4060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Ethanol absolute</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Laser microdissection system</td>
			<td style="width:148px;">Leica microsystems</td>
			<td style="width:99px;"></td>
			<td>Model AS-LMD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">DEPC</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:99px;">B22753-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bioanalyzer</td>
			<td style="width:148px;">Agilent</td>
			<td style="width:99px;"></td>
			<td>Agilent 2100 Bioanalyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Embedding mold&#160;</td>
			<td style="width:148px;">Leica Biosystems</td>
			<td style="width:99px;">14702218311</td>
			<td>6x8mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Hydrophobic barrier pen&#160;</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Frozen tissue embedding media</td>
			<td style="width:148px;">Tissue-Tek</td>
			<td style="width:99px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bioanalyzer chip</td>
			<td style="width:148px;">Aligent Technologies</td>
			<td style="width:99px;">5067-1513</td>
			<td>RNA 6000 Pico LabChip used with Agilent 2100 Bioanalyzer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Hot start reaction mix for quantitative PCR</td>
			<td style="width:148px;">Roche</td>
			<td style="width:99px;">6402712001</td>
			<td>Fast Start Essential DNA Green Master</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Reverse transcriptase</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">11752-050</td>
			<td>SuperScript III First-Strand Synthesis SuperMix for qRT-PCR</td>
		</tr>
	</tbody>
</table>

rna isolation from cell specific subpopulations using laser-capture microdissection combined with rapid immunolabeling

激光捕获显微切割（LCM）允许从薄的组织切片以高空间分辨率的特定细胞的分离。有效的LCM要求从异质组织精确识别的细胞亚群。对于LCM感兴趣细胞的识别通常是基于形态学标准或荧光蛋白记者。 LCM和快速的免疫标记的组合提供了可视化特定的细胞类型，并从周围组织分离它们的替代性和有效的手段。高质量的RNA随后可以从一个纯的细胞群中提取，并进一步处理用于下游应用，包括RNA测序，微阵列或QRT-PCR。这一方法已被预先执行，并且在几个出版物简要描述。本文的目的是要说明如何执行的细胞群体的快速免疫标记，同时保持RNA的完整性，以及如何分离使用LCM这些特定细胞。在此，我们举例说明D由免疫标记，并从1日龄的小鼠采集的多巴胺能细胞的脑组织在该多步骤过程。我们强调的是特别值得考虑的关键的关键步骤。此协议可适应各种组织和感兴趣的细胞。来自不同领域的研究人员将有可能从这种方法的演示中受益。

这种快速免疫标记过程允许同时保持RNA的完整性的关注的细胞可视化。 图1示出的RNA提取前组织制剂的步骤。 cryosectioning后，多巴胺能神经元使用针对酪氨酸羟化酶的抗体（标记的神经元出现在棕色）标记。根据实验问题，单个细胞或更大的感兴趣区域可以使用LCM被隔离（ 图1D - E）。它评估提取的RNA的质量，因为降解的RNA会对以下分析的质量相当大的影响是?...

Watch this Scientific Journal Video about RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling at JoVE.com

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

细胞在特定组织的一个子集的基因表达分析是一个重大的挑战。本文介绍了如何通过将快速免疫标记方法与激光捕获显微切割分离，从一个特定的细胞群高质量的总RNA。

从细胞亚群特异性RNA提取使用激光捕获显微切割加上快速的免疫标记

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

rna-isolation-from-cell-specific-subpopulations-using-laser-capture

Research

JoVE Journal

Biology

12.4K 次观看.  Université Laval. 细胞在特定组织的一个子集的基因表达分析是一个重大的挑战。本文介绍了如何通过将快速免疫标记方法与激光捕获显微切割分离，从一个特定的细胞群高质量的总RNA。 

视频: RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

科研

生物学

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

使用Bio - Rad公司的总RNA试剂盒从Neuroprecursor细胞的RNA提取

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

的效用舞台专用中旬至下旬<em&gt;果蝇</em&gt;卵泡隔离

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

从小鼠胰腺RNA提取:核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

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 ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

利用成熟的细胞隔离试剂盒快速分离脂滴器

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

从新鲜冷冻的小鼠骨骼中产生高质量RNA的激光捕获微解剖协议

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...