用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分
Summary
该协议描述了激光捕获微解剖，用于从小鼠胚胎的新鲜冷冻部分分离软骨和骨骼。软骨和骨骼可以通过紫罗兰染色快速可视化，并精确收集，产生高质量的RNA进行转录分析。
Abstract
激光捕获微分体（LCM）是一种强大的工具，用于从异构组织分离特定的细胞类型或感兴趣的区域。骨骼元素的细胞和分子复杂性随着发育的增加而增加。组织异质性，如软骨和骨质元素彼此或周围组织界面，是研究软骨和骨骼发育的一个障碍。我们的协议提供了一种快速的组织处理和软骨和骨分离方法，为基因表达分析产生高质量的RNA。小鼠胚胎的新鲜冷冻组织被分割，并用于用不同于周围组织的颜色来可视化软骨和骨骼的短暂克西尔紫染色。然后，幻灯片迅速脱水，软骨和骨骼随后被 LCM 分离。在此过程中，尽量减少接触水溶液，保持RNA的完整性。成功采集了小鼠Meckel在E16.5处的软骨和软骨骨，基因表达分析显示成骨细胞、成骨细胞、成骨细胞和软骨细胞的标记基因表达不同。高质量的RNA也从一系列组织和胚胎年龄中分离出来。该协议详细说明了LCM的样品制备，包括冷冻、切片、染色和脱水新鲜冷冻组织，以及LCM对软骨和骨骼的精确分离，从而产生用于转录组分析的高质量RNA。
Introduction
肌肉骨骼系统是由肌肉、结缔组织、肌腱、韧带、软骨和骨骼组成的多组分系统，由神经内膜连接，血管血管1。骨骼组织随着细胞异质性和结构复杂性的增加而发展。软骨和骨骼从相同的骨质滴激素系发展，并且高度相关。胚胎软骨和骨骼与肌肉、神经、血管和未分化的肌体结合发展。软骨也可能被骨头包围，如梅克尔的软骨和软骨在软骨内。这些组织在发育过程中通过细胞外信号在解剖学上关联并相互作用。在软骨和骨骼发育中的基因表达研究中，一个障碍是由多种组织类型组成的骨骼结构的异质性。精确分离感兴趣的特定组织是成功转录分析的关键。
激光捕获微分体（LCM）是一种强大的工具，用于分离异质组织内的细胞类型或感兴趣的区域，并且可重现，对单细胞级别2敏感。它可以精确定位和捕获感兴趣的细胞，用于转录组学、基因组学和蛋白质组学3、4中广泛的下游测定。分离的RNA、DNA或蛋白质的质量可以通过生物分析仪或等效平台进行评估。例如，RNA质量由RNA完整性数（RIN）5表示。
在这里，我们提供一个方案，通过LCM从新鲜冷冻组织中快速染色和分离软骨和骨骼。我们使用小鼠胚胎来证明该协议产生高质量的RNA，用于后续转录组分析，如RNA测序（RNA-seq）。
Protocol
根据国家卫生研究院《实验室动物护理和使用指南》，从小鼠身上获得组织，研究规程由西奈山伊坎医学院机构动物护理和使用委员会批准。 1. 新鲜冷冻标本的制备 解剖感兴趣的胚胎或组织。将样品嵌入具有最佳切削温度 （OCT） 化合物的一次性嵌入模具中。用尖或针调整试样的方向。 在干冰/甲基-2-丁烷浴中快速冷冻样品。继续执行下一步或储存在 -80 °C。<b…
Representative Results
E16.5 处新鲜冷冻小鼠组织的冠状部分用于演示 LCM 分离和收集梅克尔软骨 （MC）、软骨软骨和软骨。E16.5的小鼠胚胎被解剖，并嵌入与OCT化合物的低温模具。模具中的样品被迅速冷冻在干冰和甲基-2丁烷浴中，并储存在-80°C。 为了证明软骨和骨骼的紫紫色染色，在日冕平面上进行了冷冻切片，并在显微镜幻灯片上采集了样本。按照上述协议（步骤 3.2_3.4）对部分进行洗涤、染色…
Discussion
LCM能够从异质组织分离富集或均质细胞群。其优点包括快速、精确地捕获体内环境中的细胞，而潜在的缺点包括耗时、昂贵，且受限于用户在指定样本30中识别不同亚群的需求。该协议提供了小鼠胚胎软骨和骨骼的LCM细节，重点介绍了在快速过程中使用克西尔紫染色来可视化软骨和骨骼，从而精确收集组织，同时保持高RNA完整性，以便RNA-seq进行后续分析。该协议的一个限?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家牙科和颅面研究所（R01DE022988）和尤尼斯·肯尼迪·施莱佛国家儿童健康和人类发展研究所（P01HD078233）的支持。作者感谢生物储存库和病理学核心访问西奈山伊坎医学院的Leica LMD 6500平台。
Materials
| 2-Methylbutane | ThermoFisher Scientific | O3551-4 | |
| Bioanalyzer | Agilent | G2939BA | |
| Centrifuge tube | ThermoFisher Scientific | 339653 | Conical sterile polypropylene centrifuge tubes, 50 mL |
| Cresyl violet acetate | Sigma-Aldrich | C5042 | |
| Cryostat | Leica Biosystems | CM3050 S | |
| Delicate task wiper | ThermoFisher Scientific | 06-666 | |
| Disposable embedding mold | ThermoFisher Scientific | 1220 | |
| Distilled water | Invitrogen | 10977-015 | DNase/RNase-Free |
| Ethanol, absolute (200 proof) | ThermoFisher Scientific | BP2818 | Molecular biology grade |
| Glass PEN membrane slide | Leica Microsystems | 11505158 | |
| LCM system | Leica Microsystems | Leica LMD6500 | |
| Microscope cover glass | ThermoFisher Scientific | 12-545FP | |
| Microscope slides | ThermoFisher Scientific | 12-550-15 | |
| OCT compound | Electron Microscopy Sciences | 102094-106 | |
| PCR tube with flat cap, 0.5 mL | Axygen | PCR-05-C | LCM collection tubes |
| Permanent mounting medium | Vector Laboratories | H-5000 | |
| RNA isolation kit | ThermoFisher Scientific | KIT0204 | |
| RNase decontamination agent | Sigma-Aldrich | R2020 | RNase decontamination agent for cleaning surfaces |
| Xylene | Sigma-Aldrich | 214736 |
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Cite This Article
Wu, M., Kriti, D., van Bakel, H., Jabs, E. W., Holmes, G. Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis. J. Vis. Exp. (154), e60503, doi:10.3791/60503 (2019).