作者要感谢乔治·法尔和霍维奇实验室的成员们的有益讨论。 这项工作得到了霍华德·休斯医学研究所的支持。

这里描述的老鼠麻醉、安乐死和心脏灌注的程序是根据耶鲁大学机构动物护理和使用委员会批准的协议进行的。
1. 准备无 RNase 仪器和解决方案
<ol><li>RNase 是所有实验室表面的常见污染物，包括台面、微皮和调查器。<ol>
<li>经常更换手套或定期用 RNAse 除毒溶液擦拭。</li>
		<li>不锈钢解剖工具和玻璃器皿可在 1 小时或更> 200 °C 的加热下实现 RNase 免费。注意：蒸汽灭菌不会破坏 RNase。或者，用 RNase 除损溶液擦拭，然后用无 RNase 70% 乙醇和空气干燥。</li>
		<li>用 RNase 除毒溶液擦拭台面、其他实验室表面和微尖端，然后用无 RNase 70% 乙醇擦拭。指定用于RNA恢复和分析的特定实验室工作台或工作区域。注意：在擦拭管道管的轴之前，先取下尖端弹出器，如果可能，请将其关闭。</li>
		<li>使用经制造商认证的管子、微型中微管、移液器和微皮条尖，确保无 RNase。建议使用非常低的绑定提示和微管。如果可能，使用新打开的这些部件的盒子或袋子，并将其与一般实验室供应分开。</li>
		<li>从商业来源获得无 RNase 解决方案 [杜尔贝科的无钙和无镁 PBS ）、乙醇、无 RNase 70% 乙醇] 。每个实验使用新打开的瓶子。</li>
	</ol>
</li>
	<li>Azure B 染料的来源对于成功恢复完整的 RNA 至关重要。在将珍贵样品进行染色和收集之前，先测试每批染料。从非体验动物中收集几千个神经元，准备RNA，并确定下文所述的RNA完整性，以找到一个持续产生 RIN 数字高于 8.5 的神经元。<ol>
<li>在无 RNase 70% 乙醇中溶解 Azure B 染料 （1%，wt/vol），通常在 50 毫升离心管中溶解 30 毫升乙醇中的 0.3 克。在 RT 的摇杆上混合一夜，或直到所有粒子溶解。此解决方案的一管可用于在不影响染色强度的情况下染色多个滑梯。</li>
		<li>作为预防措施，使用不同的污渍管为每个生物复制。</li>
	</ol>
</li>
	<li>使用冷冻金和O.C.T.嵌入脐带部分，无需进一步治疗。</li>
	<li>将冷冻状态保持在 -20 至 -24 °C。 分割后，将幻灯片放在 -80 °C 冰柜中，直到准备了足够数量。幻灯片制作完后，尽快开始 RNA 准备。对于长期存储，请将未选中或部分分割的 OCT 块保持在 80 °C，而不是幻灯片。</li>
	<li>使用RNA制备套件制造商提供的解决方案，利用解剖的运动神经元制作用于RNA制备的瓜尼丁硫氰酸酯缓冲器。</li>
</ol>2. 从小鼠准备脊髓部分（图1A）
<ol><li>通过用 RNase 除损溶液烘烤或擦拭所有解剖工具、玻璃滑梯、剃须刀片和解剖平台，然后是无 RNase 70% 乙醇。让干燥2分钟。用实验室湿巾覆盖，使一切无尘。</li>
	<li>动物处理程序，包括麻醉、心脏灌注和安乐死，应按照当地机构动物护理和使用委员会（IACUC）的要求进行，目的是迅速将动物带到脊髓解剖的地步。<ol>
<li>麻醉小鼠通过内在（ip）注射致命剂量氯胺酮（450毫克/千克）。</li>
		<li>一旦达到深度麻醉，并确认缺乏脚趾捏反应，将小鼠心室侧放在解剖平台上（例如发泡胶板），打开胸腔暴露心脏，并将输液的27G蝴蝶针插入左心室（位于调查员的右侧）。使用锋利的钳子将右中庭尼克，使用围产泵以 5-7 毫升/分钟的速度与 PBS 一起香水，时间约为 2 分钟。此时，从中庭流出的液体应该基本上没有血迹。</li>
		<li>取出灌注针，斩首鼠标，并在解剖板上将其翻过来。打开皮肤露出脊柱，用小剪刀和钳子去除椎骨，抬起脊髓，同时用锋利的钳子切断神经根部。速度和实践对于从灌注开始到去除脐带至关重要：这应该不超过7分钟。</li>
		<li>在无 RNase 水中冲洗脐带 10 秒，以洗去任何残留的血液。将脊髓放在无 RNase 的玻璃滑梯上，弯曲的钳子非常温和但快速。使用干净的剃须刀刀片将脊髓横向分割成 9-10 块。</li>
		<li>将碎片放在一个充满O.C.T.的低温金中，使用相同的钳子，并用干净的针头垂直对齐它们。将模具放入含有2甲基丁烷的浅托盘中，用液氮预冷，然后将托盘放入液氮浴池中，快速冷冻脊髓片，避免RNA降解。</li>
		<li>在分割前将 OCT 嵌入式块存储在 -80 °C 中长达 6 个月。从线的检索到冻结的过程应在 5 分钟内完成，以保持 RNA 完整性。</li>
	</ol>
</li>
	<li>在-20°C的.C无鼻塞 PEN 膜 2μm 滑梯上，在 RNase 无 PEN 膜 2 μm 滑梯上，用低温统计器进行冷冻切除，产生 20 μm 切片，每个切片包含 9-10 个脊髓横截面，最初保持在室温下，以确保各部分粘附在幻灯片上。立即重新冻结冷冻器内 -20 °C 表面上的部分。将幻灯片保持在-80°C，直到使用。</li>
</ol>3. 脊髓部分污渍（图1A）
<ol><li>在干净的架子上，安排六个50毫升的圆锥形管。将所有部分填充 25-30 毫升无 RNase 70% 乙醇，以便深度足以在幻灯片浸入时覆盖幻灯片上的所有部分。制作 30-35 毫升 1% Azure B 解决方案，并将其放在同一机架上，以尽量减少洗涤或染色过程中更换溶液之间的时间。将三到四个实验室湿巾放在机架前，以便快速排出额外的溶液。</li>
	<li>将滑梯从-80°C冰柜中滑出到干冰上。将幻灯片放在戴手套的手掌上，解冻一张幻灯片。通过用实验室擦拭去滑梯上的大部分水分和凝结物，小心不要触摸部分。将滑梯放在培养皿中的新鲜硅凝胶干燥剂上30-40秒，完全干燥。如果实验室湿度高，可以在真空干燥器中进行简短的处理，以更快地干燥滑梯。</li>
	<li>洗涤和染色。<ol>
<li>将干滑梯浸入第一管无 RNase 70% 乙醇中。浸泡幻灯片30秒，然后洗滑梯，通过上下浸取45-60秒来去除OC。将滑梯从溶液中取出，将多余的液体排出实验室湿巾上。通过将幻灯片浸入第二管 70% 乙醇中，重复相同的过程。如果脊髓部分周围的 OCT 没有完全消失，则重复在第三根管中清洗。</li>
		<li>排干多余的液体后，将滑梯浸入 70% 无 RNase 乙醇中的 1% Azure B 溶液中，浸入 30-45 秒。将多余的 Azure B 排出实验室擦拭：在这一点上，整个幻灯片将是蓝色的。</li>
		<li>将滑梯浸入下一管70%乙醇中，快速上下浸3-4倍，以去除多余的染料，使特定的运动神经元染色可见。如果这些部分看起来非常暗，请重复70%乙醇的新鲜管中的脱污步骤。如果染色似乎太轻，将幻灯片返回到 Azure B 解决方案 30 秒，然后重复除污。擦去多余的乙醇，将滑梯晾干约40秒，直到所有的液体都消失。灰质为浅蓝色，而运动神经元将染成深蓝色，很容易看到。立即开始解剖。</li>
	</ol>
</li>
</ol>4. 激光捕获微分（LMD）（图1B）
<ol><li>打开显微镜并卸载管架，将 0.6 毫升微胶管的盖子连接在一起，以便收集样品。将30μl瓜尼丁硫氰酸酯裂解缓冲器放入0.6毫升微胶管的盖中。使用移液器尖端传播溶液，用液体覆盖盖住盖子表面。这样，所有收集的神经元在解剖时都会溶解在溶解溶液中。将盖子与孔和目标与显微镜软件对齐。将盖在盖上的位置，直到开始激光捕获。</li>
	<li>将空气干燥的滑梯倒置在 LMD 显微镜的舞台上，使 PEN 幻灯片的膜朝下。带部分的膜应面向孔，其下方是收集管。</li>
	<li>在开始幻灯片准备前 10 分钟打开激光。专注于具有 5 倍目标的脊髓部分。移动到 20X 目标进行解剖。<ol>
<li>用轻笔标记一个"虚拟"区域，并允许激光在不收集的情况下将其切出。这放松了 PEN 膜，并使得连续标记和切割多个区域成为可能。</li>
		<li>重新聚焦并识别心室角区域中的运动神经元。这些神经元可以通过它们的位置、金字塔形态、大尺寸和深蓝色染色（图2）来识别。</li>
		<li>使用轻笔，选择绘图工具，沿着运动神经元的边缘标记，制作一个完整的轮廓。尽量使轮廓接近运动神经元，以避免运动神经元以外的细胞污染。（事先测试某些部分，查看激光切线的宽程度，并根据需要调整间隙。切割前标记多个单元格：软件会记住的位置。</li>
		<li>单击盖位，将盖在切割位置下方。单击启动按钮，用激光启动运动神经元解剖。从单个滑动的所有部分收集所有运动神经元，进入一个微管的盖子。</li>
		<li>收集完成后，通过卸载托盘并轻轻拆下管子，将微管从支架中取出。通过上下管道将溶液完全溶解在缓冲器中的运动神经元。在 4 °C 下以 14，000 x g 的离心度将溶液移入管体 2 分钟。 立即将样品冷冻在干冰中，并将其储存在-80°C。 这个过程，从滑动染色通过运动神经元集合，应在30分钟内完成一个幻灯片，以尽量减少RNA退化。</li>
	</ol>
</li>
</ol>5. 运动神经元的RNA制备
从一种动物中解冻所有收集的神经元，并将它们汇集在一起提取RNA。样品可能看起来淡蓝色，这取决于其中运动神经元的数量（沾染了Azure B）。根据为 LMD 组织提供的协议，使用合适的套件提取 RNA，以尽可能小的体积提取。服用1微克检查样品的数量和完整性。将剩余的RNA快速冷冻在干冰上，储存在-80°C。
6. 确定RNA质量和数量
根据制造商的协议，用毛细丝电泳微流体芯片上的 1 μl 样本确定 RNA 质量。RNA 完整性编号 （RIN） 高于 8.5 的总 RNA 制剂可用于 RNA-seq 和 qRT-PCR。数据输出通常还包括样品的大致浓度。

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作者宣称他们没有相互竞争的经济利益。

通过激光微分脊髓切片产生总RNA的这一协议的成功与否的最重要指标是RIN值5。对于来自更高真核生物的RNA样本，8.5以上的值通常会产生高质量的测序和qRT-PCR数据。如果产量低但质量高，重复准备。但是，如果完整性较低（甚至为 7.5-8），则最好找到问题的根源并重试。有许多步骤可能会出问题，但实际上只有两个来源的问题 - 内源性 RNase 污染或外源 RNase 污染。第一个可以通过实践来解决，以便将鼠标从牺牲到冻结其 O .C.T 嵌入线的时间降到最低。协议中内源性 RNase 可能导致不良结果的另一点是在切片的准备过程中。每片应快速粘附在室温 PEN 滑动上，但会融化（O.C.T 变得清晰）。切片的重新冻结速度越快越好。我们使用的冷冻器在腔室内有平坦的表面，位于 -20 °C，我们用它来快速重新冻结切片。在使用的冷冻统计器中查找类似的位置，并确保切片再次迅速变得不透明。
追踪外源 RNase 的来源可能很困难，因为存在许多可能性。唯一未明确无 RNase 的试剂是 O .C.T. 和 Azure B。如果 Azure B 以前表现令人满意，请尝试一瓶新鲜的 O .C.T.，尽管这种试剂从来都不是问题。即使来自其他供应商，也可以更换无 RNase 试剂。更可能的来源是调查员。除了彻底清理工作区域外，让另一个实验室成员跟随并观察程序中的所有步骤通常也是有用的。即使这是一个人谁不定期执行这个程序，一组新的眼睛可能会拿起一个否则被忽视的污染源。
将此协议扩展到从其他脊髓细胞、来自其他物种的脊髓细胞或从其他器官的特定细胞类型恢复RNA，将需要在几个方面进行修改，旨在明确识别感兴趣的细胞，同时消除或至少尽量减少内源性RNASE的影响。在此处的协议中，快速去除和冻结脐带，加上 Azure B 染色在 70% 乙醇中，既能最大限度地减少RNA降解，又能轻松识别运动神经元细胞体。Azure B 是神经元成熟的组织化学污渍，似乎与 RNA3 结合，用于评估神经元在神经退行性疾病 6 患者脑组织解剖样本中的RNA含量。值得注意的是，Azure B 染色既不影响纯化RNA的完整性，也不影响其反向转录和分析的能力。其他染料，如紫罗兰色7 和甲苯丙胺蓝色8 已用于类似的LMD协议。在解剖细胞体时将细胞体直接收集到瓜尼丁硫氰酸酯溶液中，这提供了最终步骤，导致完整RNA的常规恢复。
其他细胞和器官可以设想类似的方法，认识到识别感兴趣的细胞，同时最大限度地减少或最好地防止RNA通过内源性RNA退化是成功分析的关键要求。在具有高水平RNASE的组织中，如胰腺，快速处理和低温已经结合，允许从β细胞中恢复RNA，利用其自然的自流可视化9。使用抗体染色进行鉴定的程序也已报告10，但尚未完全成功在我们的手中。最后，许多小鼠模型可用于在感兴趣的细胞中表达荧光融合蛋白（即GFP、YFP、RFP），这些模型可用于在LMD显微镜上的组织部分识别它们。事实上，我们用这个协议分析的老鼠菌株表达了SYD1和YFP11之间的融合蛋白，它们的脊髓运动神经元是高度荧光的。然而，我们未能找到一套乙醇洗涤和/或脱水条件，可以同时去除O.C.T.，抑制RNase活性，并持续保持足够的YFP荧光，使运动神经元可视化，并通过LMD恢复。也许可以找到一种新的荧光蛋白或在有机溶剂中保持荧光的可用蛋白质的变种，以克服这一限制。

在由多种不同细胞类型组成的哺乳动物组织中，激光捕获微分（LMD）仪器的出现为选择特定的细胞类型以进行RNA或蛋白质水平分析提供了可能性。目前，放大和下一代测序技术能够利用几千个细胞的总RNA池，对转录组进行相对彻底的清点，包括评估RNA的相对水平和识别各种拼接形式。迄今为止，蛋白质组学对几千个细胞的分析将只通过更丰富的物种进行。例如，我们从3000-4000个运动神经元细胞体中鉴定出1000种最丰富的蛋白质中的 <1000种（未显示），朱和同事最近报告说，他们从15，000个肿瘤细胞1中识别出2，665种蛋白质。然而，随着质谱学的进一步发展，这种分析很可能将扩展到更深的深度。
在这里，我们提出了一个特定的协议，用于从小鼠脊髓运动神经元细胞体的LMD，然后准备RNA。该协议用于比较来自预症状转基因突变分解（SOD）1肌萎缩侧索硬化（ALS）小鼠运动神经元的RNA，这些小鼠的RNA由RNA-seq和qRT-PCR验证2从野生型SOD1转基因菌株中制备。值得注意的是，运动神经元，在脊髓灰质前角，占细胞总数的<10%，被星形细胞的海洋包围，因此，它们的转录轮廓不能轻易地从整个线的研究中去分解。因此，激光捕获方法非常适合分析这些细胞中的RNA表达，通过在短暂的内皮盐水灌注后快速切除和冷冻脐带来保持生理学。运动神经元somata是大，因此很容易检测，在这里使用一种染料，具有很强的亲和力神经元3。此外，在这样的大小下，这些细胞为捕获的每个细胞提供相对较大的RNA。所使用的程序，如下所述，可以很容易地调整，以获得其他神经元细胞类型，以及潜在的其他细胞类型，具体识别通过染料染色技术或抗体染色。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Ketamine</td>
			<td style="width:195px;">Webster Veterinary</td>
			<td style="width:155px;">26637041101</td>
			<td style="width:339px;">Any source approved by the institutional veterinary service</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Dissection tools (scissors, forceps,&#160;etc.)</td>
			<td></td>
			<td></td>
			<td style="width:339px;">Any convenient source of high-quality dissection instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Cryostat&#160;</td>
			<td>Leica Microsystem</td>
			<td>CM3050S</td>
			<td style="width:339px;">Other cryostats should be suitable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">LMD6000 B</td>
			<td>Leica Microsystem</td>
			<td></td>
			<td style="width:339px;">Other LMD systems have not been tested</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">RNase-free PEN-membrane 2 &#956;m slides&#160;</td>
			<td>Leica Microsystem</td>
			<td>11505189</td>
			<td style="width:339px;">Other slide types have been tested and do not perform well</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:268px;">RNeasy Micro kit (50)</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td style="width:339px;">Any kit that uses guanidine thiocyanate lysis buffer and has been tested for recovering small quantities of RNA can be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Agilent 2100 BioAnalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2939A</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Azure B Certified&#160;</td>
			<td>MP Biomedicals</td>
			<td>190520</td>
			<td style="width:339px;">Critical! Product from any source has to be tested before use for its ability to maintain RNA integrity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">PBS</td>
			<td>Sigma Life Science</td>
			<td>D8537</td>
			<td style="width:339px;">Any reliable source of RNase-free PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">RNA Ethanol 70% (made with DEPC treated water)</td>
			<td>American Bioanalytical</td>
			<td>AB04010-00500</td>
			<td style="width:339px;">Any reliable source, can be lab-made</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Water, RNase-free, Endotoxin tested (treated with DEPC to eliminate RNase)</td>
			<td>American Bioanalytical</td>
			<td>AB02128-00500</td>
			<td style="width:339px;">Any reliable source, can be lab-made</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">RNase AWAY</td>
			<td>Invitrogen Life Technologies</td>
			<td>10328-011</td>
			<td style="width:339px;">Other similar RNase removal reagents, such as RNaseZAP (also from Invitrogen) can be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">2-Methylbutane&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>78-78-4</td>
			<td style="width:339px;">Any source</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Maxymum Recovery microfuge tubes (0.6 and 1.5 ml) and pipette tips&#160;</td>
			<td>Axygen&#160;</td>
			<td>311-03-051 &#38; 311-09-051</td>
			<td style="width:339px;">Ultra-low retention, RNase free tubes and tips; test other manufacturer&#39;s products before using</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">O.C.T. Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Cryomold Intermediate Size</td>
			<td>Tissue-Tek</td>
			<td>4566</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Lab wipes (Kimwipes)</td>
			<td>KIMTECH</td>
			<td>34155</td>
			<td style="width:339px;"></td>
		</tr>
	</tbody>
</table>

production of rna for transcriptomic analysis from mouse spinal cord motor neuron cell bodies by laser capture microdissection

从感兴趣的细胞中制备高质量的RNA对于精确和有意义地分析细胞类型之间、健康和疾病中同一细胞类型之间的转录差异或遵循药理治疗至关重要。在脊髓中，运动神经元的这种制备，是许多神经和神经退行性疾病感兴趣的目标，由于运动神经元占细胞总数的<10%，因此变得复杂起来。激光捕获微分（LMD）已经开发出来来解决这个问题。在这里，我们描述了一个协议，快速恢复，冻结，并部分鼠标脊髓，以避免RNA损伤的内源性和外源性RNA，然后沾上Azure B在70%乙醇，以确定运动神经元，同时保持内源性RNASE抑制。然后，LMD 用于将染色的神经元直接捕获到瓜尼丁硫氰酸酯裂解缓冲器中，保持 RNA 完整性。标准技术用于恢复总RNA并测量其完整性。然后，此材料可用于 RNA-seq 和 qRT-PCR 对成绩单的下游分析。

上面概述的协议和 图1 应产生50 ng或以上的总RNA与RIN的 >rin 8.5从3，000-4，000脊髓运动神经元细胞体解剖从9-10 20 μm切片约20笔幻灯片。由于此幻灯片数量小于鼠标脊髓 O .C.T.嵌入块的 10%，因此可以返回存储在 -80 °C 的块，并切开更多切片以进行另一轮 RNA 制备。如果分析需要大量的RNA，最好在几天内通过解剖和RNA准备一次只制作幻灯片（如20张幻灯片）的数量，然后为第二轮制作更多的幻灯片并组合产品。请务必先检查每个准备的RIN，以避免将好的与坏的结合在一起。RNA-seq 方法正变得越来越敏感，新的 PCR 技术有望比现有方法具有更高的灵敏度。因此，需要更少的神经元细胞体的RNA，从而允许更大的测序深度和更全面的验证有趣的命中。另一方面，完全遵守 MIQE 关于 qRT-PCR 分析和验证4 的建议可能需要更大的金额，以便对低丰度RNA做出必要的控制反应。
图 2 显示了 Azure B 染色和解剖后脊髓心室角部分的典型图像系列。在面板A中，大而暗的彩色细胞体是运动神经元，通过抗聊天抗体染色2得到证实，并且很容易与较小的相邻细胞区别。面板 B 显示与用轻笔勾勒出的单独细胞体的相同区域，并按显微镜软件编号。轮廓紧贴细胞体的边缘，激光的切割宽度稍小。此间距必须确定为每个激光功率设置，以尽量减少无关材料的包含，同时避免运动神经元细胞索的损失。面板 C 和 D 显示激光切割后的部分，单个细胞体已落入收集盖。请注意，切割幅度并不完全遵循面板 C 中的轻笔轮廓，但基本上不位于它之外。这种不规则性在面板 D 中很明显，每个切口周围的黑暗区域也很明显，这反映了激光对组织的热损伤。因此，如果两个细胞体非常接近对方，最好勾勒出它们进行单次切割的轮廓，而不是试图单独切割它们，并失去一些RNA，以在它们近接触的区域造成热损伤。
图3 显示了从LMD收集的约4，000个小鼠运动神经元细胞体中准备的RNA样本的RNA完整性的电泳分析的典型结果。上面板是总RNA的1μl产生的电图：右侧的插图是凝胶的图像。在这两个，请注意突出的核糖核化物峰值。下面板是包含RNA大小标准的车道的电传。分析软件计算出此样本的RIN为 9.8，浓度为 4.9 ng/μl。分析后，共提供了 13 μl 的此解决方案，为下游 qRT-PCR 验证成绩单金额提供了 64 ng 的 RNA。对于中等丰富的成绩单的典型 qRT-PCR 分析，总 RNA 的 0.15-0.20 ng 足以产生准确的量定量 2，因此此制备提供的 RNA 量足以验证多个引物集的多个脚本，如建议的那样 4。当然，大量的输入RNA可用于验证罕见的成绩单。这一数额也足以允许图书馆的编写和下一代RNA-seq。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51168/51168fig1highres.jpg" src="/files/ftp_upload/51168/51168fig1.jpg"> 图1。 脊髓切片生产概况和脊髓运动神经元细胞体激光捕捉微分。A） 切片生产和染色的主要步骤。B） 脊髓部分和激光解剖的卡通视图。 <a href="https://www.jove.com/files/ftp_upload/51168/51168fig1highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51168/51168fig2highres.jpg" src="/files/ftp_upload/51168/51168fig2.jpg"> 图2。Azure B 染色运动神经元细胞体激光解剖的前后图像。A） 染色部分的腹角的一部分。注意四个大的，深色的细胞。也有一些轻微的染色和稍小的细胞，它们不是运动神经元（通过抗聊天抗体染色确认;未显示，但见Bandyopadhyay2），不会选择解剖。许多小的黑点可能是神经元过程（树突和轴突），但这还没有得到证实。B） 用轻笔勾勒出四个细胞体。请注意，轮廓紧贴细胞的边距，它们之间的距离最小。此间距应为每个显微镜和激光建立经验。该软件对单个神经元进行编号，从而简化了对已收集到的神经元的跟踪。C 和 D） 激光切割后，包含细胞体的碎片已落入收集帽。请注意，留下的孔比轮廓稍大，且不规则。这反映了激光束的宽度及其微变化的切割效率，具体取决于组织的局部组成。同样明显的是，由于激光造成的局部加热损伤，每个孔周围暗组织的边缘。
<a href="https://www.jove.com/files/ftp_upload/51168/51168fig2highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51168/51168fig3highres.jpg" src="/files/ftp_upload/51168/51168fig3.jpg"> 图3。从LMD样品中对RNA完整性的电光分析。A） 通过微流体芯片上的微胶囊电泳分析，从上述协议从约 4，000 个运动神经元细胞体中制备的 RNA 的 1μl 等音标进行了分析。显示电球图，并显示突出的核糖核化物峰值。右边是凝胶本身的图像。B） 显示尺寸标准的电图，右侧有相应的凝胶通道。仪器软件计算出此样本的RIN为9.8，浓度为4.9 ng/μl。 <a href="https://www.jove.com/files/ftp_upload/51168/51168fig3highres.jpg" target="_blank"></a>

Watch this Scientific Journal Video about Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection at JoVE.com

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

通过激光捕捉微分，用 Azure B 染色 70% 乙醇的脊髓部分后，从小鼠脊髓运动神经元的细胞体中制备了高质量的总RNA。从 3，000-4，000 个运动神经元中恢复足够的 RNA（+40-60 ng），以便通过 RNA-seq 和 qRT-PCR 进行下游 RNA 分析。

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 ...

production-rna-for-transcriptomic-analysis-from-mouse-spinal-cord

Research

JoVE Journal

Neuroscience

10.6K 次观看.  Yale School of Medicine. 通过激光捕捉微分，用 Azure B 染色 70% 乙醇的脊髓部分后，从小鼠脊髓运动神经元的细胞体中制备了高质量的总RNA。从 3，000-4，000 个运动神经元中恢复足够的 RNA（+40-60 ng），以便通过 RNA-seq 和 qRT-PCR 进行下游 RNA 分析。

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

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

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

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

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

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  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

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

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

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

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

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 ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

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

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

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

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

了解运动神经元对脊髓刺激的反应

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

作者聚焦：推进脊髓刺激 - 通过膜片钳电生理学探索运动神经元的细胞反应 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...