应用激光微分射发现人体肾脏组织中的区域转录学
June 9th, 2020
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。
激光显微切割允许对肾组织样本中空间定义的区域进行转录组学分析。这使我们能够有独特的机会来识别感兴趣的结构，即使在疾病引起的变化发生之后也是如此。该方法的主要优点是与其他组学技术（如单细胞 RNA 测序）互补。
它提供了显著的组织经济性，甚至允许检测低表达的转录本。采集组织样本后，使用设置为零下 20 摄氏度的低温恒温器将样本切割成 12 微米的厚度，并使用载玻片适配器将样本固定在专门的激光显微切割载玻片上。在冷冻切片后 10 天内，将 89.2 微升不含 RNase 的 10% BSA 的 PBS 溶液与 4 微升 FITC 鬼笔环肽、1.5 微升 DAPI、2 微升直接偶联 Alexa Fluor 546 的目标抗体和 3.3 微升 RNase 抑制剂混合。
在零下 20 摄氏度的 100% 丙酮下洗涤玻片 1 分钟，然后将玻片放入湿度室中。用新鲜的不含 RNase 的 PBS 洗涤玻片顶部两次，每次洗涤 30 秒，然后用 10% BSA 的无 RNase PBS 洗涤两次，每次 30 秒。最后一次洗涤后，在室温下用制备好的抗体溶液处理样品 5 分钟，然后用 10% BSA 的无 RNase PBS 再洗涤 2 次。
第二次洗涤后，在组织培养罩中将玻片风干 5 分钟，然后将玻片加载到激光显微切割平台上。将适用于 PCR 工作的高压灭菌的 500 μL 微量离心收集管安装到设备上，其中包含来自 RNA 分离试剂盒的 50 μL 提取缓冲液。上样后，通过染色、形态和位置鉴定样品中感兴趣的区域。
使用免疫荧光勾勒出至少 500， 000 微米见方的感兴趣片段。然后启动激光显微切割，使用大于 40 的激光功率切除该区域。激光显微切割过程完成后，盖上微量离心机收集管并用力轻弹管，以确保内容物从瓶盖移动到管底。
离心后，将试管在 42 摄氏度的水浴中孵育 30 分钟，然后再次离心试管。然后将上清液从零下 80 摄氏度储存到新的 500 微升试管中。肾脏中肾小管亚段的鉴定是通过对独特的肾小管标志物以及细胞形态和结构标志物进行抗体染色来完成的。
荧光标记与形态学组织特征和图像结构的空间定位相结合，有助于以高度的置信度可视化肾脏亚段。在这种具有代表性的下游测序分析中，满足最小解剖区域起始量的成功率大于 90%，导致可用于基因检测的总 mRNA 数量很高。在此分析中，100% 的样品满足至少 10， 000 个基因的最低检测阈值。
激光显微切割后，可以将已知标记物的基因表达的富集分析与其他隔室的基因表达进行比较。在该分析中，通过激光显微切割区域转录组学鉴定了每个亚段的一个标记物，该转录组学还产生了相应肾单位亚段的特异性免疫组织化学染色。需要注意的是，激光显微切割在很大程度上依赖于用户的专业知识来精确识别要解剖的感兴趣结构。
该技术允许研究特定的区域基因表达特征，这些特征可用于评估参与疾病进展的潜在途径，以及健康组织的生物学特性。
View the full transcript and gain access to thousands of scientific videos
概述
本研究提出了一种用于人类肾脏组织特定亚段（包括肾小球和近端小管）激光显微解剖的协议。从这些部分分离出来的RNA能够进行转录组分析，以揭示与不同肾脏结构相关的基因表达模式变化。
关键研究组成部分
研究领域
- 转录组分析
- 肾脏生物学
- 疾病进展调查
背景
- 研究肾脏亚段的重要性
- 分析疾病后空间定义结构的挑战
- 与其他组学技术的互补性
使用的方法
- 激光显微解剖
- 人类肾脏组织样本
- RNA分离和测序
主要结果
- 解剖和RNA恢复的高成功率
- 肾脏亚段基因表达标记的鉴定
- 展示了与疾病进展相关的潜在通路
结论
- 该协议展示了一种研究肾脏区域基因表达的有效方法。
- 研究结果有助于理解肾脏生物学和潜在的疾病机制。
常见问题
Executive Industry Relevance
Laser microdissection enables spatially resolved transcriptomic profiling of human kidney tissue, addressing a critical gap in discovery biology where dissociation-based methods lose anatomical context. This approach supports target validation by linking gene expression signatures to defined nephron sub-segments, enhancing mechanistic de-risking in renal disease models. The method provides predictive confidence for pathway interrogation and portfolio prioritization in early discovery workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by mapping gene expression to anatomically defined kidney compartments.
- Operational Value: Supports biological de-risking through spatially anchored transcriptomic signatures that maintain tissue context.
- Predictive Value: Facilitates target confidence by correlating regional marker expression with functional sub-segment identity.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream omics workflows by isolating high-integrity RNA from microdissected regions.
- Operational Value: Ensures assay standardization and reproducibility through QC parameters assessing specimen adequacy and dissection success rates.
- Scalability: Enables reliable compound evaluation by providing quantitative, spatially resolved transcriptomic outputs for screening campaigns.
Translational & Preclinical Research
- Translational Continuity: Connects discovery findings to preclinical validation through disease-relevant system modeling using human kidney sub-segments.
- Biomarker Alignment: Supports translational biomarker development by identifying compartment-specific gene expression profiles detectable in tissue.
- Risk-Adjusted Advancement: Informs preclinical decision-making by revealing pathway activity in anatomically precise disease-affected regions.
Pipeline & Workflow Integration
Laser microdissection fits within the discovery continuum from hypothesis testing to lead identification, enabling spatially resolved molecular profiling that bridges imaging and omics.
- Discovery Biology: Supports hypothesis testing and pathway clarification by linking gene expression to structural landmarks in kidney tissue.
- Screening: Delivers assay readiness through high-success-rate dissection (>90%) and sufficient RNA yield for sequencing applications.
- Analytics: Provides quantitative dependent variable measurements (gene detection thresholds ≥10,000 genes) enabling cross-condition comparison.
- Translational Research: Advances preclinical continuity by preserving spatial context lost in dissociation-based methods.
- Enterprise Reuse: Functions as a reusable capability across tissue types via antibody marker substitution, supporting platform-wide adoption.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity in renal target validation.
- Operational Value: Enhances standardization and reproducibility through defined workflow parameters and QC checkpoints.
- Strategic Value: Improves go/no-go decisions by enabling risk-adjusted prioritization based on sub-segment-specific biology.
- Portfolio Impact: Supports capital efficiency through early de-risking of renal targets using spatially resolved evidence.
Implementation Considerations
- Requires expertise in immunofluorescence staining and morphological identification of renal sub-segments.
- Dependent on laser microdissection platforms and RNA isolation kits compatible with low-input samples.
- Necessitates cross-team standardization for staining protocols, dissection thresholds, and QC metrics.
- Adaptation across models requires validation of antibody markers and structural landmarks in target tissues.
- Practical limitations include user-dependent precision in region identification and RNA integrity maintenance during processing.
