Name: application of laser microdissection to uncover regional transcriptomics in human kidney tissue
Uploaded: 2020-06-09T14:30:00.000+00:00
Duration: 5 min 46 s
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，将 1.2 μm LMD PPS-膜（聚（苯乙烯硫化物）滑入紫外线（在组织培养层流罩中）30 分钟。将幻灯片存储在室温下，以获得最佳的组织粘性。</li>
	<li>将低温静电冷却至-20°C。 清洁工作表面并安装新的切割刀片。</li>
	<li>将一个小滑动盒（用 RNase 表面净化溶液清洗）放在冷冻机房内，用新鲜切割的组织存储幻灯片。</li>
	<li>将 OCT 中的标本粘附在组织支架上，并允许其平衡几分钟，以达到室温，并加强 OCT 块与持有人之间的粘附。使用热提取器帮助该过程。</li>
	<li>将标本切成 12μm 的厚度，并使用滑动适配器将其贴在专用 LMD 幻灯片上。每张幻灯片每张幻灯片可容纳一个肾切除部分，或每张幻灯片最多可容纳两个肾脏活检部分。将滑梯存放在 -80 °C 下，并配有干燥盒，并存放在紧闭的塑料袋内，以防止滑动盒内积聚多余的水分。</li>
	<li>用标本 ID、日期和幻灯片编号标记每个幻灯片。</li>
	<li>在冷冻分离初始日期的 10 天内将幻灯片与标本一起使用。</li>
</ol>2. 激光微分
<ol><li>染色前立即在无 RNase PBS 中准备 10% BSA 中的抗体混合 （Ab-Mix），添加以下信息:FITC-Phalloidin 的 4μL， 1.5 μL 的 DAPI、2 μL 的 Tamm-Horsfall 蛋白 （THP） 抗体直接与亚历克萨氟 546、3.3 μL 的 Rnase 抑制剂和 89.2 μL 的 10% BSA 在 PBS 中（达到 100μL 的体积）。 注:替代抗体可用于代替THP抗体。例如，2 μL 的兆林/LRP2 抗体，直接与 Alexa Fluor 568 结合，可用于标记近焦管。Ab-Mix 包含 LRP2 或 THP 抗体（分别可视化近体管或粗升肢）。其他抗体可根据用户需求进行验证。</li>
	<li>在冰冷（-20°C）100%丙酮中清洗滑梯1分钟，并将其移动到湿度室。</li>
	<li>用无 Rnase PBS 清洗幻灯片顶部 30 s。 重复。</li>
	<li>在无 Rnase Pbs 中用 10% 的 Bsa 清洗幻灯片顶部 30s。 重复。</li>
	<li>应用 Ab 混合 5 分钟。</li>
	<li>在无 Rnase Pbs 中用 10% 的 Bsa 清洗幻灯片顶部 30s。 重复。</li>
	<li>空气干燥滑梯5分钟，并加载到激光微散射切割平台上。</li>
	<li>安装适合 PCR 工作的集合管（自动切口 0.5 mL 微中心管），包含 50 μL 的 RNA 隔离套件中的提取缓冲区。</li>
	<li>继续进行LMD。在最多 2 小时内完成每个 LMD 会话。<ol>
<li>收集LMD前后免疫荧光图像，使用显微镜摄像头验证解剖的操作间变异性以及档案目的、培训和执行协议的质量评估。为了获得 0.5 – 1 ng 的RNA，至少需要 500，000 μm2 区域。这通常需要使用高达 8 x12 μm 厚的部分，以获得足够数量的材料，供所有感兴趣的子部分使用。</li>
		<li>通过染色、形态和位置来识别感兴趣的区域，并使用大于 40 的激光功率将其除名。 注:以下是解剖标准。近脉管由 FITC-法洛丁和 LRP2 标签定义。厚升肢由 THP 标记定义。收集管道由核形态 （DAPI） 定义，没有其他污渍。球菌由FITC-法洛丁和形态学定义。间质被定义为染色管之间的区域。</li>
	</ol>
</li>
	<li>通过将两个 12 μm 部分贴在 LMD 幻灯片上并将整个部分解剖成提取缓冲区，获得批量横截面表达签名。</li>
	<li>LMD 流程完成后，关闭收集的微中心管并大力轻弹，以确保内容从盖子移动到管子底部</li>
	<li>将管子以 3，000 x g 为 30 s 提供离心机。</li>
	<li>将管子浸泡在 42 °C 水浴中 30 分钟。</li>
	<li>将管子在 3，000 x g 下离心 2 分钟。</li>
	<li>将超强元转移到一个新的0.5 mL管，并将其存储在-80°C中。</li>
</ol>3.RNA隔离
注:对于此RNA隔离协议，我们已从商用RNA隔离套件中修改了协议。制造商的协议已修改，以满足为该项目设置的质量控制要求。
<ol><li>在每个RNA净化柱 （PC） 中添加 250 μL 的有条件缓冲区 （CB），并在室温下孵育 5 分钟。</li>
	<li>离心所有 PC 1 分钟，在 16，000 x g。</li>
	<li>将 50 μL 的 70% 乙醇（在 Kit 中提供）加入带组织样本的管子中。通过上下管道将样品混合好。不要漩涡。不要离心。</li>
	<li>将混合物在 100 x g（ 绑定 RNA）下将混合物转移到有条件的 PC 和离心机中 2 分钟，在 16，000 x g 下快速跟随离心 30 秒（以消除流经）。重复此步骤，如果超过 1 管组织样本可用于任何给定的子段。</li>
	<li>在 8，000 x g时，将 100μL 的洗涤缓冲区 1 （WB1） 添加到 PC 和离心机中 1 分钟。</li>
	<li>每个样品准备 40 μL 的 DNase（将 5 μL 的 Dnase 添加到 35μL 的 RDD 缓冲区）。然后直接在PC膜上加入40微升的混合物，并在室温下孵育15分钟。</li>
	<li>将 40 μL 的 WB1 添加到 PC 的膜上，在 8，000 x g下将离心机加到 15 s。</li>
	<li>在 PC 膜上添加 100 μL 的洗涤缓冲区 2 （WB2），在 8，000 x g下将离心机加 1 分钟。</li>
	<li>在PC膜上加入100μL的WB2，离心机2分钟，在16，000 x g，紧接着在16，000 x g下离心1分钟。</li>
	<li>将PC转移到一个新的0.5 mL管。</li>
	<li>在膜上加入 12 μL 的 Elution 缓冲区 （EB），并在室温下孵育 7 分钟。因此，所有汇集解剖组织样本的最终体积为每子段 12 μL。</li>
	<li>将样品在1000 x g 下抽取1分钟，然后在16，000 x g下抽取2分钟。</li>
	<li>将 2 μL 转移到用于生物分析器分析的新鲜管中（以防止冻结事件）。</li>
	<li>将所有管子存放在-80°C中，直到准备好进一步处理。</li>
</ol>4. RNA 测序
<ol><li>使用商用生物分析仪和用于测量少量RNA的芯片评估每个样本的质量，用于测序。</li>
	<li>需要在库准备和测序之前遵循质量控制 （QC） 参数:散装 RNA 大于 4 ng，每个子段大于 0.5 - 1 ng:超过 200 核苷酸 （DV200） 的抄本百分比对于 LMD 标本（最佳 =75%）要求大于 25%。</li>
	<li>使用商业 cDNA 库准备套件进行图书馆准备，用于少量退化 RNA。对于补充中列出的商业套件，我们建议使用选项 2，这需要至少 DV200 的 25%，并且没有碎裂。某些测序技术可能需要更高的最低 DV200 阈值，例如 30%7。</li>
	<li>添加 cDNA 适配器和索引。</li>
	<li>使用磁珠技术净化RNAseq库。</li>
	<li>在 RNAseq 库放大步骤之前，使用商用 rRNA 去除套件耗尽核糖体 cDNA。</li>
	<li>使用磁珠技术以 2 ng/μL cDNA 库浓度净化最终的 RNAseq 库。</li>
	<li>在商业测序系统上执行 75 bp 配对端的 RNA 测序，批量读取/示例为 3000 万次读取/示例，子细分部分执行 1 亿次读取/示例。</li>
	<li>使用参考RNA（25微克）与每次测序运行，以允许控制批次效果。我们的参考RNA的初始浓度为1微克/μL，而测序中使用的最终浓度为25 ng/μL。</li>
	<li>利用 FastQC 应用程序进行数据分析，以评估测序、间源读取和线粒体读取的质量，并确定归因于基因的读数。</li>
	<li>使用集成基因组学查看器 （IGV） 进行对齐和边缘 R/rbamtool 以进行成绩单表达测量。</li>
	<li>删除读数少于 100，000 的样本。在筛选出用户定义阈值下表达的低表达基因后，Quantile 使数据集中的原始读数规范化。</li>
	<li>将表达式量化为兴趣子细分与所有其他子细分的平均值和 log2 转换的比率。同一基因的相对表达可以比较子段和样本:然而，由于基因和RNA物种之间潜在的退化差异，比较两种不同基因之间的相对表达并不理想。</li>
	<li>进行浓缩分析，比较特定于每个肾素子段的一组制造商的基因表达，同时对参考RNA样本进行分批比较。R值>0.9平均表达式1个标准偏差范围内的批量效应被认为是可以接受的。更高的批次效果分数需要额外的规范化。任何偏离接受批次效应的运行都可以标记。每次测序运行的 Q30 应大于 +90%。</li>
</ol>

<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>

作者没有什么可透露的。

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 &amp;micro;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&amp;amp;D Systems</td><td>AF5144</td><td>Directly conjugated to Alexa Fluor 546</td></tr><tr><td>Tissue-Tek&amp;reg; 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 ...

application-laser-microdissection-to-uncover-regional-transcriptomics

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

3.8K 次观看.  Indiana University School of Medicine. 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。

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

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 ...

科研

神经科学

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

小鼠肾中微RNA表达与单体尿阻的定量实时PCR评价

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

遗传学

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

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

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

发育生物学

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

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

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

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

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, ...

生物学

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

人类前列腺上皮的激光捕获显微切割的RNA分析

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

医学

Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a significant approach to understanding the fundamental physiological and molecular processes of renal pathophysiology. The methods and protocols we present here will allow for the molecular dissection of the kidney in each specific region of interest related to disease sequelae. To determine the effects of disease on specific kidney regions and structures with unique functions, the goals of this protocol are to demonstrate simplified mouse kidney compartmentalization and renal cortical tubule isolation techniques in tandem with streamlined label-free quantitative proteomic workflows. Combining these methods will assist in the identification of perturbed molecular patterns in the whole kidney, medullary compartments, and cortical tubule structures of kidneys, with the ultimate and eventual goal of single-cell proteomics in pathological contexts. Applying these methods in virtually any disease model will be helpful in delineating mechanisms of pathology related to kidney dysfunction.

Here, we present a detailed protocol for proteomic analysis of the whole kidney, isolated cortical tubule, and medullary proteomes. The study also compares regional proteomes in a diabetic mouse model and non-diabetic mice.

小鼠肾损伤糖尿病发病机制中全肾、髓质和皮质小管的比较蛋白质组学分析

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a ...

生物化学

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

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

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

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

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

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

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

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 ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

应用激光微分射发现人体肾脏组织中的区域转录学

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