Name: a laser capture microdissection protocol that yields high quality rna from fresh-frozen mouse bones
Uploaded: 2019-09-16T15:00:00
Description: Watch this Scientific Journal Video about A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones at JoVE.com

作者感谢乌特·泽茨和尼科尔·金纳的出色技术支持，以及Vetcore和动物护理人员的支持。

严格按照现行的动物护理指南使用小鼠的骨组织，并竭尽全力将动物的痛苦降到最低。
1. 动物和冷冻嵌入
<ol><li>室内动物在恒定室温（RT;24°C）和12小时光/12小时暗循环条件下，可免费获得食物和水。 注：本研究中的骨组织取自3个月大的雄性野生型C57BL/6小鼠。</li>
	<li>在尸检时，在一般麻醉下从腹腔中分离小鼠（氯胺酮/西拉辛，100/6毫克/千克腹腔内）。 <s...

<ol>
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I. <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:+Insights+into+Methods+and+Applications.">Laser Capture Microdissection: Insights into Methods and Applications.</a> Laser Capture Microdissection: Methods and Protocols. , 1-17 (2018).</li><li>Burgemeister, R., Gangnus, R., Haar, B., Sch&#252;tze, K., Sauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+quality+RNA+retrieved+from+samples+obtained+by+using+LMPC+(laser+microdissection+and+pressure+catapulting)+technology.">High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology.</a> Pathology, Research and Practice. 199 (6), 431-436 (2003).</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>Cl&#233;ment-Ziza, M., Munnich, A., Lyonnet, S., Jaubert, F., Besmond, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+RNA+during+laser+capture+microdissection+by+performing+experiments+under+argon+atmosphere+or+using+ethanol+as+a+solvent+in+staining+solutions.">Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.</a> RNA. 14 (12), 2698-2704 (2008).</li><li>K&#246;lble, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LEICA+microdissection+system:+design+and+applications.">The LEICA microdissection system: design and applications.</a> Journal of Molecular Medicine. 78 (7), B24-B25 (2000).</li><li>B&#246;hm, M., Wieland, I., Sch&#252;tze, K., R&#252;bben, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbeam+MOMeNT:+non-contact+laser+microdissection+of+membrane-mounted+native+tissue.">Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue.</a> The American Journal of Pathology. 151 (1), 63-67 (1997).</li><li>Micke, 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+fluid+cover+medium+provides+superior+morphology+and+preserves+RNA+integrity+in+tissue+sections+for+laser+microdissection+and+pressure+catapulting.">A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting.</a> The Journal of Pathology. 202 (1), 130-138 (2004).</li></ol>

RNA质量和数量在样品制备的所有阶段（如组织操作、LCM 过程和RNA提取）都会受到负面影响。因此，开发了LCM协议，以获得足够数量的高质量RNA，用于后续的基因表达分析。
对于LCM，大多数实验室使用7~8微米厚的2节。较厚的部分将允许收获更多的材料。然而，如果它们太厚，这可能会降低显微分辨率，激光可能无法穿过。最佳组织厚度可能取决于所使用的LCM系统（以?...

组织由异构和空间分布的细胞类型组成。给定组织中的不同细胞类型对同一信号的反应可能不同。因此，能够分离特定的细胞群，以评估不同细胞类型在生理和病理条件下的作用是至关重要的。激光捕获微解剖（LCM）提供了一种相对快速和精确的方法，用于从复杂组织1中分离和去除指定的细胞。LCM 系统使用激光束的力量将感兴趣的细胞与组织组织部分分离，而无需酶处理或培养生长。这意味着细胞处于其自然组织栖息地，并且组织架构，包括不同细胞之间的空间关系被保留。捕获的细胞和残余组织的形态保存完好，可以从同一张幻灯片按顺序取样几个组织成分。分离的细胞随后可用于对其RNA、DNA、蛋白质或代谢物含量2、3的后续分析。
为了分析不同细胞群的基因表达，或经过不同的处理，有必要获得足够质量和数量的mRNA，以便随后的分析4，5。与DNA相比，RNA对固定更敏感，当目标是研究RNA时，建议使用冷冻组织。由于 mRNA 因无处不在的核糖核酸酶 （RNase） 而迅速降解，因此在试样处理和制备过程中需要严格的无RNase条件，并避免在室温下储存样品。此外，快速技术没有任何长时间的水相步骤是至关重要的，以防止RNA降解6。RNA的产生和完整性也会受到LCM过程和LCM系统使用7，8的影响。目前，有4个不同操作原理的LCM系统可供2。RNA提取方法也很重要，因为不同的RNA分离试剂盒已经过测试，RNA数量和质量有显著差异，7，8。
任何组织制备方法都需要在获得良好的形态对比和保持RNA完整性之间找到平衡，以便进行进一步分析。为了从骨头中制备冷冻部分，开发了一个粘合膜，并持续改进了9。骨部分被切割并直接在粘结膜上染色。这种胶膜适用于多种染色类型，并可用于使用LCM9、10、11、12、13分离感兴趣的细胞和骨低温细胞。 14.所有步骤，包括手术切除，嵌入，冻结，切割和染色可以在不到一小时的时间内完成。重要的是，细胞，如成骨细胞，骨衬里细胞，和成骨细胞可以清楚地识别9，10，11，12，13，14。该方法具有快速简便的优点。生成骨骼冷冻节的另一种方法是使用磁带传输系统15。然而，后一种技术更耗时，需要额外的仪器，因为部分必须通过紫外线（UV）交联将部分从胶带转移到预涂膜幻灯片上。虽然磁带传输系统已成功与 LCM16、17、18、19结合，但需要注意的是，交联涂层可以创建背景模式，可以干扰细胞类型识别20。
通常，只有少量的RNA从微解剖细胞中提取，RNA的质量和数量通常通过微毛细血管电泳21来评估。计算机程序用于为称为RNA完整性数（RIN）的RNA提取物分配质量指数。RIN值1.0表示完全退化的RNA，而值10.0表示RNA完全完好22。通常，超过5的索引被认为足以用于RNA研究。据报道，RIN值为5.0±10.0的样本中的基因表达模式相互关联良好23。虽然这种方法的灵敏度很高，但由于仅能检测到总RNA的50 pg/μL，但如果样品中的RNA浓度非常低，则很难获得质量评估。因此，为了评估RNA的质量，LCM后剩余的组织部分常被用来提取RNA，通过移液缓冲液移至幻灯片24。
虽然LCM已广泛用于不同的冷冻组织，但提取RNA的RIN值很少报告。此外，没有比较研究来阐明研究小鼠骨骼RNA的最适当方法。在本研究中，使用成年小鼠股骨的冷冻部分优化样品制备、LCM协议和RNA提取，以获得高质量的RNA。本协议特别针对使用重力采集样品的LCM系统进行了优化。

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>63689-25ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol EMPLURA</td>
			<td>Merck Millipore</td>
			<td>8,18,76,01,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive film (LMD film)</td>
			<td>Section-Lab</td>
			<td>C-FL001</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer System</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico Chip Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus HistoGene Staining Solution</td>
			<td>Applied Biosystems</td>
			<td>12241-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryofilm fitting tool</td>
			<td>Section-Lab</td>
			<td>C-FT000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat Leica CM 1950</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass microscope slides, cut colour frosted orange</td>
			<td>VWR Life Science</td>
			<td>631-1559</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology tissue molds PVC</td>
			<td>MEDITE</td>
			<td>48-6302-00</td>
			<td></td>
		</tr>
		<tr>
			<td>LMD7&#160;Laser&#160;Mikrodissektion&#160;System</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low profile Microtome Blades Leica DB80 XL</td>
			<td>Leica Biosystems</td>
			<td>14035843496</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>VWR Life Science</td>
			<td>E476-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PET membrane slides 1.4 mircon</td>
			<td>Molecular Machines &#38; Industries GmbH</td>
			<td>50102</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away surface decontaminant</td>
			<td>Molecular BioProduct</td>
			<td>7002</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek optimal cutting temperature (OCT) compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR Life Science</td>
			<td>2,89,73,363</td>
			<td></td>
		</tr>
	</tbody>
</table>

a laser capture microdissection protocol that yields high quality rna from fresh-frozen mouse bones

RNA的产生和完整性对RNA分析起决定性作用。然而，在整个激光捕获微解剖（LCM）过程中，保持RNA完整性往往在技术上具有挑战性。由于LCM研究对低含量材料起作用，因此对RNA产量有限的担忧也很重要。因此，开发了一种LCM协议，以获得足够数量的高质量RNA，用于骨细胞的基因表达分析。评估了染色方案、冷冻切片厚度、微解剖组织数量、RNA提取试剂盒和LCM系统对从微解剖骨细胞中获得的RNA产量和完整性的影响。八微米厚的冷冻骨部分使用胶膜制成，并使用商业LCM染色的快速协议进行染色。样品夹在聚乙烯对苯二甲酸酯（PET）膜和粘合膜之间。利用重力采集样品和基于柱的RNA提取方法获得足够产量的高质量RNA的LCM系统。目前的研究集中在小鼠股骨部分。然而，这里报道的LCM协议可用于研究任何硬组织细胞在生理条件和疾病过程中的原位基因表达。

开发了一种LCM协议，以获得足够数量的高质量RNA，用于小鼠股骨细胞的基因表达分析。在优化的协议中，8 μm厚的冷冻骨部分被切割在粘结膜上，并使用商业LCM冷冻截面染色的快速协议进行染色。样品夹在PET膜和粘合膜之间。小鼠骨细胞使用使用重力采集样品的LCM系统进行微解剖。采用基于柱的RNA提取方法获得足够产量的高质量RNA。使用微毛细血管电泳测量了分离RNA的产量和完整性（<strong class="xf...

Watch this Scientific Journal Video about A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones at JoVE.com

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

开发了激光捕获微分体（LCM）协议，以获得足够数量的高质量RNA，用于骨细胞的基因表达分析。目前的研究集中在小鼠股骨部分。然而，这里报道的LCM协议可用于研究任何硬组织细胞的基因表达。

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 ...

你有没有想过利用激光捕获微分裂的力量来分析自然环境中特定骨细胞中的基因表达？在这里，我们描述了一个协议，将使您能够从小鼠骨骼的冷冻获得足够数量的高质量RNA。这项技术是研究基因工程小鼠模型或疾病模型中特定骨细胞中基因表达变化的重要工具。此处描述的协议侧重于小鼠骨骼，但可用于就地研究任何物种中任何硬组织的细胞中的基因表达。帮助证明这个程序，将是克里斯蒂安舒勒，一个技术人员从我的实验室。在全身麻醉下通过排泄对小鼠进行安乐死后，迅速切除整个股骨。使用手术刀和纸巾清洁周围软组织的股骨。接下来，将最佳切削温度化合物倒入嵌入模具中，并将股骨放入嵌入模具的底部。在液氮中捕捉冻结样品。样品完全冷冻后，用铝箔包装，然后放在干冰上转移到冰柜中。将它们存放在零下80摄氏度，直到准备进一步处理。首先，将低温恒温设置为零下19摄氏度，将块架的温度设置为零下17摄氏度。接下来，用70%乙醇擦拭低温恒温器的内部。将硬组织、玻璃滑梯和安装工具的一次性刀片放入低温恒温器中冷却，并在截切期间将它们放在低温恒温器内。将干冰上冷冻组织块转移到低温恒温器，并允许其平衡至少 10 分钟。然后，按嵌入模具的底部将 OCT 块从模具中推出。将足够的 OCT 介质涂抹到块架上，将块粘附到其上，并等待 OCT 介质完全冻结。在此之后，将块架放在物体支架中并拧紧到位。调整刀片位置，以 15 微米切割增量修剪块，以去除覆盖样品的 OCT。调整低温恒温器生成八微米的截面，并切割两到三个将被丢弃的低温分段。将粘合膜放在块上，并使用安装工具将薄膜粘附在块上。现在，以恒定的速度缓慢地进行切割，同时通过薄膜按住该部分。将样品朝上放在低温恒温器内低温杆上预冷玻璃滑梯上的薄膜，以避免样品解冻。使用胶带将薄膜固定到玻璃滑梯上，以便于染色。在烟气罩中，准备必要的乙醇、无 RNase 水和冰上二甲苯的解决方案，如文本协议中所述。在95%乙醇中孵育部分30秒。然后，小心地将部分浸入无 RNase 水中 30 秒，以完全去除 OCT。接下来，将50微升商业 LCM 冷冻部分污渍分配到该部分，并在室温下孵育 10 秒。将幻灯片的边缘放在吸水纸巾上，将滑轨排空。用 100%乙醇冲洗部分 30 秒，以去除多余的污渍。将骨骼部分浸入含有100%乙醇的第二管中30秒，然后将它们转移到100%二甲苯30秒。将胶粘膜放在干玻璃滑梯上作为支撑，注意尽可能平整薄膜。在此之后，将 PET 膜框架幻灯片放在薄膜上，然后短暂地将戴手套的手指压在薄膜上，将其连接到薄膜上。样品将夹在膜和胶粘膜之间。胶粘膜不应折叠或起皱，薄膜和膜之间不应有气泡。首先使用表面除敏剂清洁舞台端盖支架。将幻灯片加载到滑动架中，将盖盖装入盖座。接下来，调整焦点并获取具有 1.25 倍目标的幻灯片概览。更改为 40 倍目标并调整焦点。使用幻灯片概述，选择感兴趣的区域。然后，调整此处所示的激光参数，确保针对每个目标优化这些参数。如果激光无法切割样品，请增加激光功率。根据形态学标准，选择胸膜或皮质骨骼中的成骨细胞、骨质细胞和骨衬细胞。为远离目标细胞的激光路径绘制一条线，以尽量减少紫外线激光的伤害。如果激光未能切割样品，请多次应用激光或检查目标是否切割不全的斑点，并使用移动和切割选项切割这些点的组织。将每种细胞类型收集在一个单独的0.5毫升管盖中。首先，将含有β甲醇的50微升解液分配到收集管的盖子中。将样品在盖子上上下一分钟，将样品上下打上。接下来，向下旋转裂解液，并将含有β-美甲乙醇的裂解缓冲液的00微升添加到管子中。对于每张幻灯片，准备一个标有微离心管，其中含有含有β-β甲醇的解液缓冲液的350微升。使用 LCM 后剩余的部分提取 RNA。小心地将薄膜与膜分离，然后缓慢地将解解缓冲液%地将样品解到该部分上。然后，将酸盐样品放在干冰上，储存在零下80摄氏度。准备好继续时，在室温下解冻落酸。在此之后，将收集管中 LCM 收获的细胞中的 lysates 转移到新的 1.5 毫升微离心管中，然后根据制造商的说明提取 RNA。在这项研究中，开发了一种LCM协议，以获得足够数量的高质量RNA，用于小鼠股骨骨细胞的基因表达分析。LCM 使用 LCM 系统执行，该系统使用重力收集样品。使用微胶囊电泳测量分离RNA的产率和完整性。使用不同解解方案获得的RNA质量和数量的差异，可以在代表性的凝胶和电谱图中看到。当样品在盖上上下下点一分钟时，可以从一平方毫米的微分受损骨组织中分离出约8.5毫微克RNA。RIN 值为 8.60。或者，LCM 使用 LCM 系统执行，该系统使用散焦激光脉冲，将材料弹射到悬垂胶盖中。对于新鲜冷冻的骨骼，可以从一平方毫米的微分受损骨组织中分离出大约1.6毫微克RNA。RIN 值为 1。总之，在此过程中要记住的最重要的事情是正确应用站立协议，防止三明治复合物中部分完全干涸，使用适当的 LCM 系统，并且不超过收获细胞的时间限制。从捕获的细胞中分离出RNA后，您可以使用RNA进行表达分析，例如通过RNA寻寻。最后，我们想提醒您，二甲苯和β-甲苯甲醇是需要用引擎盖处理的危险物质。此外，在处理液氮和低温叶片时，请采取适当的预防措施。

Research

JoVE Journal

Biology

Laser Capture Microdissection of Mammalian Tissue

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

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

人死后脑组织中的单细胞群获得高品质的RNA

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

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 ...

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

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 ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

一个网络工具，用以生成高质量机器可读的生物学途径

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

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 ...