Name: optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue
Uploaded: 2018-06-14T14:30:00
Description: Watch this Scientific Journal Video about Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue at JoVE.com

我们感谢捐助者及其家人使这项工作成为可能。我们还感谢国际医学研究所和田纳西州捐助者服务机构的工作人员协助协调本研究所用组织的收集工作。这项工作得到了国家卫生研究院、nih OT2-OD023850 到 EMS2的赠款和 nih T32-DK007673 向湄京集团提供的助学金支持。我们感谢范德比尔特翻译病理学共享资源的工作人员获得 LCM 仪器和关于组织准备的建议。范德比尔特组织病理共享资源部分由 NIH 赠款 P30-CA068485-14 和 U24-DK059637-13 支持。我们感谢华盛顿大学医学院遗传学系的基因组技术访问中心, 以帮助进行基因组分析。GTAC 的部分支持的癌症中心支持补助金 #P30 CA91842 Siteman 癌症中心和信息和通信技术/CTSA 赠款 #UL1TR002345 从国家研究资源中心 (NCRR), 一个组成部分的国家卫生研究院 (nih) 和 nih医学研究的路线图。这份出版物完全是作者的责任, 不一定代表 NCRR 或 NIH 的官方观点。

此处描述的所有协议都已由范德比尔特大学机构审查委员会批准。
1. 组织到达前的准备工作
<ol><li>获得适当的 IRB 批准, 并与人体器官捐赠机构协调, 从捐赠者那里获得新切除的无固定肠道组织, 以满足研究所需的所有研究标准。 注: 本协议可适用于小鼠和其他啮齿动物模型使用。<ol>
<li>在每肠段切除后立即, 用冷冻 PBS 或组织贮存培养基彻底冲洗腔体, 以除去残留的腔内材?...

<ol>
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PALM Protocols - RNA handling Available from: <a target="_blank" href="https://hcbi.fas.harvard.edu/files/zeiss_labprotocol_rna_0811.pdf">https://hcbi.fas.harvard.edu/files/zeiss_labprotocol_rna_0811.pdf</a> (2011)</li><li>. Workflows &amp; Protocols: How to Use a Leica Laser Microdissection System and Qiagen Kits for Successful RNA Analysis Available from: <a target="_blank" href="https://www.leica-microsystems.com/science-lab/laser-microdissection/workflows-protocols-how-to-use-a-leica-laser-microdissection-system-and-qiagen-kits-for-successful-rna-analysis/">https://www.leica-microsystems.com/science-lab/laser-microdissection/workflows-protocols-how-to-use-a-leica-laser-microdissection-system-and-qiagen-kits-for-successful-rna-analysis/</a> (2015)</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arcturus+HistoGene+Frozen+Section+Staining+Kit:+User+Guide.">Arcturus HistoGene Frozen Section Staining Kit: User Guide.</a> Biosystems, A. , (2010).</li><li>Cummings, M., 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+robust+RNA+integrity-preserving+staining+protocol+for+laser+capture+microdissection+of+endometrial+cancer+tissue.">A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue.</a> Analytical Biochemistry. 416 (1), 123-125 (2011).</li><li>Grover, P. 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这个过程使许多肠道神经节的有效收集成为 rna 的来源在这里, 我们加速了现有协议中概述的过程, 同时最大限度地保留了 RNA 完整性。由于这一程序中的所有步骤都是相互依存的, 因此必须在研究开始时消除所有的问题, 并尽可能以同样的方式编写所有的样本, 以获得可靠的 RNA--
从程序的开始, 如果时间间隔 (冷缺血时间) 在器官捐赠时组织的收集和接收组织在实验室中的处理时...

本方法旨在利用激光捕获显微切割技术从人体肠道组织获得高质量的肠道神经节 RNA 样品。此处描述的协议已经过优化, 以提供足够的 rna 质量和 rna 排序 (rna 序列) 的产量, 并打算用于与新鲜切除, 无固定, 闪光冷冻人类肠道组织。
功能性胃肠道和肠道运动障碍影响美国每四人之一。肠神经系统, 也称为第二脑1, 往往是这些疾病的中心, 因为它在肠道动态平衡和运动中起着关键的作用。对肠道运动的操纵一般限于手术切除 aganglionic/noncontractile 组织, 慢性饮食修饰和/或药物。令人惊讶的是, 成人的完整转录仍然是排序, 大大限制了我们的能力, 以鉴定在体内的分子, 可以被靶向药用或用于干细胞治疗。
从人肠道神经节中分离 RNA 的方法相对较少。第一种方法, 细胞离解2, 需要高的孵化温度和长时间的潜伏期;这两种都是已知的促进 RNA 降解和改变转录2,3。另一种方法, LCM, 更可靠地保存转录和保护 RNA 完整性。虽然一些研究已经使用 LCM 收集神经节从新鲜冷冻人类肠道组织4,5,6, 这些方法要么是由于 RNA 质量和数量低下, 是相当劳动密集型的, 或需要修改染色或 RNA 提取技术, 在我们的手工作。为保存在 lcm 产品手册中发现的 RNA 而设计的其他 lcm 协议提供了额外的改进7、8, 但在应用于肠神经节8的隔离时需要适应,9. 出于这些原因, 我们开发了一种基于这些资源的优化协议, 它从人类肠道神经节产生大量的高完整性 RNA, 具有相对较快的工作流, 并在大型样本数。
在本研究中, 我们提出了一个优化程序的概要, 以促进分离高完整性的 RNA 从肠道神经节从切除人肠道组织。我们的方法包含五重要方面。首先, 新切除的, 不固定的人体肠道样本应该修剪到大小, 所有多余的水分去除与实验室组织和扁平化在一个大的基础模具之前, 在干冰的泥浆和 2-丁烷 (2 MB) 的闪光冻结。其次, 肠道组织学切片应准备获得肌间丛丛在一张幻灯片上的完整平面, 它提供了一个大的肠道神经节的负载。这个步骤的成功在很大程度上取决于组织的准备过程。第三, 存在的神经节的不均匀结构要求使用聚乙烯 napthalate (钢笔) 膜幻灯片6, 这在 LCM 过程中提供了最大的速度和精度。第四、乙醇相容染料, 如甲酚紫, 应用于保存 RNA 完整性, 同时染色肠神经节。最后, rna 提取过程对于一个成功的 rna 序列的结果是至关重要的。我们寻求一种 rna 提取方法, 产生高 rna 完整性, 最大限度地提高 rna 的产量, 当开始与小收集肠道神经节, 消除 DNA 污染, 并保留尽可能多的 rna 物种。
同时, 本研究对这些因素的优化, 大大加快了肠道神经节的工作流程和产量, 具有异常的 RNA 数量和质量。结果在相当大的样本群中大致一致, 表明了这种方法的一致性。此外, 我们已经使用这些方法成功地序列了许多来自肠道神经节的 RNA 样本。这里所强调的策略也可以广泛地适应于对周围和中枢神经系统所期望的神经节或细胞核进行 LCM, 以及其他需要隔离高质量 RNA 的情况。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">10% Neutral-buffered formalin</td>
			<td style="width:280px;">Sigma</td>
			<td style="width:131px;">HT501128-4L</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Methylbutane</td>
			<td>Fisher</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-mL syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mL conical tubes, polypropylene&#160;</td>
			<td>Corning</td>
			<td>05-526B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Base molds 37x24x5mm, disposable</td>
			<td>Electron Microscopy Sciences</td>
			<td>5025511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Belzer UW&#160; Cold Storage Solution</td>
			<td>Bridge to Life</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CapSure Macro LCM caps</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cling Wrap, Press&#8217;n Seal&#160;&#160;</td>
			<td>&#160;Glad&#160;&#160;</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cresyl Violet Acetate</td>
			<td>Acros Organics</td>
			<td>AC405760025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cutting Board</td>
			<td>Electron Microscopy Sciences</td>
			<td>63308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 190-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 200-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Microscope slides</td>
			<td>Fisher</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gloves, extended cuff</td>
			<td>Microflex&#160;</td>
			<td>19010144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gowns, surgical-disposable</td>
			<td>Kimberly-Clark&#160;</td>
			<td>19-088-2116</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tray, 20 L (large polypropylene sterilizing pan)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>1335920B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (large)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (small)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser Capture Microdissection System (ArcturusXT)</td>
			<td>ThermoFisher</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica Cryostat Chuck, large,&#160; 40 mm&#160;</td>
			<td>Southeast Pathology Instrument Service</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light Microscope</td>
			<td>Olympus</td>
			<td>CX43</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (20X)</td>
			<td>Olympus UPlanFl 20X/0.50, &#8734;/0.17</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (10X)</td>
			<td>Olympus UPlanFl 10X/0.25, &#8734;/-</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Sieves</td>
			<td>Acros Organics</td>
			<td>AC197255000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease-free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PicoPure RNA extraction kit</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pellet Pestle</td>
			<td>Kimble Kontes</td>
			<td>4621973</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEN membrane LCM slides</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-free tubes, 1.5 mL</td>
			<td>Ambion</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZAP&#160;</td>
			<td>Sigma</td>
			<td>Sigma-R2020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;A&#34; (PicoPure)</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;B&#34; (RNeasy&#160; Micro kit)</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Method &#34;C&#34; (TRIzol)</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;D&#34; (RNeasy PLUS Mini kit)</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splash Shield, disposable faceshield</td>
			<td>Fisher</td>
			<td>17-310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors, Surgical, 14.5 cm &#34;sharp-sharp&#34;</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, cellulose acetate (0.2 &#956;m)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>190-2520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Freezing Medium</td>
			<td>General Data Healthcare</td>
			<td>TFM-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylenes</td>
			<td>Fisher</td>
			<td>X3P1GAL</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue

该方法的目的是通过激光捕获显微切割 (LCM) 从未固定的、新鲜切除的人肠道组织采集的肠神经节获得高完整性的 RNA 样本。我们已经确定了工作流程中的五个步骤, 对于从肠神经节获取 rna 分离的关键, 它的质量和数量足以为 rna--首先, 在准备肠道组织时, 每个样品必须有所有多余的液体去除的印迹之前, 使浆膜尽可能多地在底部的大基模具。然后在干冰和2丁烷的泥浆上迅速冻结样品。第二, 当切片的组织, 重要的是放置 cryomolds, 使肠道部分平行的全平面的肌间丛丛, 从而产生最大的肠道神经节每张幻灯片的面积。第三, 在 LCM 过程中, 聚乙烯 napthalate (笔) 膜幻灯片在收集肠神经节时, 可以提供最大的速度和灵活性来勾勒出肠道神经节的非均匀形状。第四, 对于不同的肠道神经节可视化, 与乙醇相容的染料, 如甲酚紫罗兰, 提供优良的保存相对于水染料的 RNA 完整性。最后, 为了从捕获的神经节中提取 rna, 我们观察了商业 rna 提取试剂盒中产生的 rna 数量和质量的差异, 同时消除了 DNA 污染。这些因素在当前协议中的优化极大地加速了工作流, 产生了异常的 RNA 质量和数量的肠神经节样本。

我们已经对现有的协议进行了几项改进, 通过 LCM 能够相对快速地从人体肠道样本收集肠道神经节, 达到 RNA 序列的标准。首先, 我们优化了在 2 MB (图 1B) 放置在干冰泥浆表面的大型基模中肠道组织段的快速冻结。最佳的成功在 cryosectioning 和随后 LCM 是通过放置肠段平在一个基地模子, 面对黏膜边上升 (图 1 b-1 c) 获得。确保基础...

Watch this Scientific Journal Video about Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue at JoVE.com

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

该协议的目的是获得高完整性的 RNA 样本从无固定的, 新鲜切除的人肠道组织, 利用激光捕获显微切割 (LCM) 分离。该协议包括制备人体肠道组织、cryosectioning、乙醇染色和脱水、LCM 和 RNA 提取的闪光冷冻样品。

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元：一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

从开发小脑显微切割分离的不同的细胞群

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...