Name: application of laser microdissection to uncover regional transcriptomics in human kidney tissue
Uploaded: 2020-06-09T14:30:00
Description: Watch this Scientific Journal Video about Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue at JoVE.com

一般： 作者要感谢肾脏精密医学项目（www.kpmp.org）的研究人员给予的亲切支持和建议。
资金： 国家卫生研究院/NIDDK K08DK107864（M.T.E.）为这项工作提供了支持：尼赫/尼德克UG3DK114923（T.M.E.，P.C.D.）：R01DK099345（T.A.S.）。这份手稿中报道的研究得到了国家糖尿病和消化和肾脏疾病研究所（NIDDK）肾脏精密医学项目（KPMP）（www.kpmp.org）的支持，获奖号码为U2CDK114886。
数据和材料可用性： 数据存档在基因表达综合（GEO #待定）

这项研究被印第安纳大学机构审查委员会（IRB）批准使用。
注：使用此协议与肾脏肾切除组织（高达2厘米的X和Y尺寸）保存在最佳切割温度（OCT）化合物，并存储在-80°C。 以限制RNA污染的方式执行所有工作，使用干净的一次性手套和面罩。确保所有表面的清洁。优化此协议的设备是具有脉冲紫外激光的激光微发射系统。
1. 冷冻剖射
<ol><li>在低温之前，...

<ol>
	<li>El-Serag, H. B., 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=Gene+expression+in+Barrett's+esophagus:+laser+capture+versus+whole+tissue.">Gene expression in Barrett's esophagus: laser capture versus whole tissue.</a> Scandinavian Journal of Gastroenterology. 44 (7), 787-795 (2009).</li><li>Kohda, Y., Murakami, H., Moe, O. W., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+segmental+renal+gene+expression+by+laser+capture+microdissection.">Analysis of segmental renal gene expression by laser capture microdissection.</a> Kidney International. 57 (1), 321-331 (2000).</li><li>Murakami, H., Liotta, L., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IF-LCM:+laser+capture+microdissection+of+immunofluorescently+defined+cells+for+mRNA+analysis+rapid+communication.">IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication.</a> Kidney International. 58 (3), 1346-1353 (2000).</li><li>Woroniecki, R. P., Bottinger, E. P. <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+of+kidney+tissue.">Laser capture microdissection of kidney tissue.</a> Methods in Molecular Biology. 466, 73-82 (2009).</li><li>Noppert, S. J., Eder, S., Rudnicki, M. <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+of+renal+tubule+cells+and+linear+amplification+of+RNA+for+microarray+profiling+and+real-time+PCR.">Laser-capture microdissection of renal tubule cells and linear amplification of RNA for microarray profiling and real-time PCR.</a> Methods in Molecular Biology. 755, 257-266 (2011).</li><li>Amini, 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=An+optimised+protocol+for+isolation+of+RNA+from+small+sections+of+laser-capture+microdissected+FFPE+tissue+amenable+for+next-generation+sequencing.">An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.</a> BMC Molecular Biology. 18 (1), 22 (2017).</li><li>. Catalytic FFPE Nucleic Acid Isolation for best NGS Performance Available from: <a target="_blank" href="https://celldatasci.com/products/RNAstorm/RNAstorm_Technical_Note.pdf">https://celldatasci.com/products/RNAstorm/RNAstorm_Technical_Note.pdf</a> (2016)</li><li>Micanovic, R., Khan, S., El-Achkar, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+laser+micro-dissection+of+specific+nephron+segments+in+the+mouse+kidney+allows+targeted+downstream+proteomic+analysis.">Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis.</a> Physiological Reports. 3 (2), (2015).</li><li>Lake, B. B., 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+single-nucleus+RNA-sequencing+pipeline+to+decipher+the+molecular+anatomy+and+pathophysiology+of+human+kidneys.">A single-nucleus RNA-sequencing pipeline to decipher the molecular anatomy and pathophysiology of human kidneys.</a> Nature Communications. 10 (1), 2832 (2019).</li><li>Rodriguez-Canales, J., 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=Optimal+molecular+profiling+of+tissue+and+tissue+components:+defining+the+best+processing+and+microdissection+methods+for+biomedical+applications.">Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.</a> Methods in Molecular Biology. 980, 61-120 (2013).</li><li>Hipp, J. D., 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=Computer-Aided+Laser+Dissection:+A+Microdissection+Workflow+Leveraging+Image+Analysis+Tools.">Computer-Aided Laser Dissection: A Microdissection Workflow Leveraging Image Analysis Tools.</a> Journal of Pathology Informatics. 9, 45 (2018).</li></ol>

基于LMD的转录学是一种有用的技术，将基因表达锚定在组织内的特定区域。这项技术的基础及其在肾脏中的潜在应用以前曾被描述过8。然而，针对下游RNA测序的高精度解剖，荧光解剖的优化、现代化和精简并不普遍。由于这种方法在组织内具有空间基础，因此有可能揭示组织结构中新颖或被低估的标记物，尤其是在疾病状态下。事实上，细胞的许多常见标记可能随疾病而改变，...

研究组织标本的技术进步增进了对各种器官健康状况和疾病状况的了解。这些进展突出表明，病理学可以在有限的区域或特定的细胞类型开始，但对整个器官有重要影响。因此，在当今的个性化医学时代，了解细胞和区域层面的生物学非常重要，而不仅是全球的生物学。这在肾脏中尤为明显，它由各种专门的细胞和结构组成，这些细胞和结构对病理应激有微分的启动和/或反应。各类人类肾脏疾病的发病机理仍不为人所知。生成一种方法来研究人类肾脏间质的特定管状部分、结构或区域的基因表达变化，将增强发现区域特定变化的能力，这些变化可能告知疾病的发病机制。
人类肾脏活检标本是一种有限而宝贵的资源。因此，应优化肾组织转录经济学的技术，以节约组织。在细胞和区域一级研究转录学的现有方法包括单细胞RNA测序（scRNaseq）、单核RNAseq（snRNaseq）、原位空间杂交和激光微分（LMD）。后者非常适合精确隔离组织部位内感兴趣的区域或结构，用于下游RNA测序和分析2、3、4、5。LMD 可在解剖过程中使用荧光成像，依靠基于验证标记的特定细胞类型或结构进行识别。
激光微分解剖辅助区域转录学的独特特征包括：1） 保存细胞和结构的空间背景，补充单细胞技术，即通过表达而不是组织学识别细胞：2） 该技术因抗体标记定义表达特征而通知并受到其他成像技术的通知：3） 即使在疾病标记发生变化时识别结构的能力：4） 在大约20，000个基因中检测低表达的记录：和5）显着的组织经济性。该技术可扩展至肾脏活检，其核心厚度小于 100μm，是获取足够的RNA所必需的，并且能够使用存档的冷冻组织，这些组织通常可在大型存储库或学术中心6.
在随后的工作中，我们详细描述了区域和批量转录经济学技术，并优化了一种新的快速荧光染色方案，用于人体肾脏组织。这种方法改进了经典的LMD探索，因为它为间质和肾上分段提供了单独的表达数据，而不是聚合的管间表达。其中包括为确保严格性和可重复性而实施的质量保证和控制措施。该协议使细胞和感兴趣的区域可视化，从而从这些孤立区域令人满意地获取RNA，从而允许下游RNA测序。

<table>
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>270725-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure Beads</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>2100</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccant Cartridge</td>
			<td>Bel-Art</td>
			<td>F42046-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>RDD buffer is included in the pakage</td>
		</tr>
		<tr>
			<td>Laser Microdissection Microscope</td>
			<td>Leica</td>
			<td>LMD6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Megalin/LRP2 Antibody</td>
			<td>Abcam</td>
			<td>ab76969</td>
			<td>Directly conjugated to Alexa Fluor 568</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>ThermoFisher</td>
			<td>AB-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica</td>
			<td>DFC700T</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (RNAse Free)</td>
			<td>VWR</td>
			<td>K812-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin (Oregon Green 488)</td>
			<td>ThermoFisher</td>
			<td>O7466</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>PPS-membrane slides</td>
			<td>Leica</td>
			<td>11505268</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR Human Reference Total RNA 25 &#181;g</td>
			<td>Takara Clontech</td>
			<td>636690</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Eukaryote Total RNA Pico Chip</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Away</td>
			<td>ThermoFisher</td>
			<td>7000</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Inhibitor</td>
			<td>ThermoFisher</td>
			<td>AM2696</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencer (HiSeq or NovaSeq)</td>
			<td>Illumina</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTer Stranded Total RNAseq Pico Input v2</td>
			<td>Takara Clontech</td>
			<td>634411</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamm-Horsfall Protein Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF5144</td>
			<td>Directly conjugated to Alexa Fluor 546</td>
		</tr>
		<tr>
			<td>Tissue-Tek&#174; O.C.T. Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of laser microdissection to uncover regional transcriptomics in human kidney tissue

人体肾脏组织的基因表达分析是了解平衡和疾病病理生理学的重要工具。需要提高这项技术的分辨率和深度，并将其扩展到组织内的细胞水平。虽然单核和单细胞RNA测序的使用已变得普遍，但从组织分离中获得的细胞的表达特征并不维持空间背景。基于特定荧光标记的激光微散射 （LMD） 将允许在已知本地化下分离特定结构和感兴趣的细胞组，从而能够在肾脏组织中获取空间锚定转录特征。我们优化了LMD方法，以快速荧光染色为指导，分离出人类肾脏内的五个不同的隔间，并从有价值的人类肾脏组织标本进行后续RNA测序。我们还提供质量控制参数，以便评估所收集标本的充分性。本手稿中概述的工作流程显示了这种方法的可行性，即高度自信地分离子段转录签名。此处介绍的方法也可用于替代相关抗体标记的其他组织类型。

样品
我们提供来自9个参考肾切除术（在印第安纳大学获得的3个标本和通过肾脏精密医学项目获得的6个标本）的数据，利用快速荧光染色方案分离肾肾部分和间歇区域。在这个过程中使用的部分是从已故的肾脏捐献者或未受影响的肿瘤肾切除术中获得的。这些样本没有病理学证据，如从邻近部分（图1A）采集的H&E污渍所示。
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Watch this Scientific Journal Video about Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue at JoVE.com

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

我们描述了人类肾脏的子段的激光微分射方案，包括球状、近肌管、厚升肢、收集管道和间质。然后从获得的隔间中分离RNA，进行RNA测序，以确定每个子段内转录签名的变化。

应用激光微分射发现人体肾脏组织中的区域转录学

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...

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