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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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 thématique de recherche en Neurosciences (CTRN). HDB is funded by a scholarship from the Fonds de Recherche en Santé du Québec (FRSQ) and ML is a FRSQ Chercheur-Boursier.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Membrane coated slides | Leica Biosystems | 11505158 | MembraneSlides PEN-Membrane 2,0 µm; 50 pcs |
| Surface RNAse decontamination solution | Life technologies | AM9780 | RNaseZap |
| Fixative | 70% Ethanol kept at -20 °C | ||
| Anti-TH | Pel-Freez | P40101 | 1:25 |
| RNAse free buffer | DEPC PBS + 1% BSA+0.02% triton | ||
| RNAse inhibitor | Promega | N2615 | Rnasine Plus Rnase Inhibitor 40 kU/ml (stock) |
| Anti-rabbit biotinylated | Vector Laboratories | BA-1000 | |
| Vectastain Elite ABC kit Standard | Vector Laboratories | PK-6100 | 8µl/ml |
| DAB Peroxidase Substrate Kit | Vector Laboratories | SK-4100 | |
| RNA isolation kit | Life technologies | KIT0204 | Arcturus PicoPure RNA Isolation Kit |
| H2O2 30% | Sigma | HC4060 | |
| Ethanol absolute | |||
| Laser microdissection system | Leica microsystems | Model AS-LMD | |
| DEPC | Alfa Aesar | B22753-14 | |
| Bioanalyzer | Agilent | Agilent 2100 Bioanalyzer | |
| Embedding mold | Leica Biosystems | 14702218311 | 6x8mm |
| Hydrophobic barrier pen | Vector Laboratories | H-4000 | |
| Frozen tissue embedding media | Tissue-Tek | 4583 | |
| Bioanalyzer chip | Aligent Technologies | 5067-1513 | RNA 6000 Pico LabChip used with Agilent 2100 Bioanalyzer |
| Hot start reaction mix for quantitative PCR | Roche | 6402712001 | Fast Start Essential DNA Green Master |
| Reverse transcriptase | Life technologies | 11752-050 | SuperScript III First-Strand Synthesis SuperMix for qRT-PCR |
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