激光捕捉显微解剖对甲醛固定小鼠肝组织用于RNA分析
2026年4月17日
本协议描述了一种利用激光捕捉显微解剖，从对甲醛固定、最佳切割温度化合物嵌入小鼠肝脏组织的特定组织学区域中分离RNA的方法，实现针对性的下游基因表达分析。
我们的研究主要探讨慢性肝病背景下的肝细胞相关病理生理。该方案使肝组织切片中的细胞分离和RNA提取成为可能。与FFPE（对甲醛固定或CT协议）不同，采用保存和迭代，能够进行精确的微观讨论和高质量样品，用于后续OMI分析。
开始时，430分钟后将笔膜玻片暴露在紫外线下。在玻片上涂上0.1毫克每毫升聚L赖氨酸，然后自然风干。用中性安装介质封住每个滑套的边缘。
使用RNase去污剂，擦拭冷冻容器及所有工具的所有可接触表面。使用75%乙醇。彻底清洁刷子和工具。
现在，将低温容器温度设为零下20摄氏度，让它稳定下来。从零下80摄氏度的储存中取回肝脏样本。将样品放入低温室至少15分钟以达到平衡。
在样品台上涂抹一层2至3毫米厚的OCT化合物。然后将样品台放置在快速空闲的架子上或低温箱内，预先凝固化合物层。在预固化层上涂抹少量OCT化合物后，将平衡的组织块水平放置于该化合物上，并保持10到15秒。
然后将带有组织块的样品台安装到低温屏样品头上。现在小心地将刀片插入低温容器一侧的夹板和背板之间。夹紧夹紧杆以固定刀片。
将低温箱厚度设置调到10微米。将组织切成切片，直接安装在低温室内预处理的笔膜载玻片上，温度保持在零下20摄氏度。将玻片在室温下解冻20分钟。
用RN ACE无添加PBS洗玻片三次，每次洗410秒。将过滤后的Mayer血液素溶液加入组织切片，浸泡5到10秒。然后用无核酸酶水洗三次载玻片，每次10秒，以去除残留的血液素。
用显微镜评估染色质量。用吸水纸吸干玻片，然后完全自然风干。用RNase消毒剂擦拭实验室工作台表面。
打开控制器，让显微镜完成初始化。将按键顺时针从关闭位置旋转到开启位置以启动激光系统。打开电脑，启动激光捕捉微型解剖软件。
然后等待初始化完成。现在将一根无菌的0.2毫升PCR管插入采集装置。使用移液管向PCR管盖加入10微升蛋白酶K消化缓冲液。
将采集装置装入显微镜标本架。在找零收集器设备窗口里。去调整参考点，调整相机焦点以清晰看到参考点，必要时用箭头控制来居中。
将组织切片装入样本架，组织朝下，标签位置在右侧。选择10倍物镜，识别具有特定病理特征的关注区域。进入激光菜单。
点击校准，选择“是”，完成校准。在激光控制面板中，将激光功率设为50，光圈为5，速度为9。然后用图形工具栏在目标区域周围画一个圆。
选择单一形状，按下开始切割键进行激光捕捉显微解剖，并定位采集设备以采集解剖组织。要捕捉文档和图片，选择文件并选择保存图片以首选格式保存前后图片。收集完所需电池后，点击卸载键将收集器从舞台上移除。
确认盖子内仍有10微升蛋白酶K消化缓冲液，必要时补充。关闭管子，彻底旋转。然后在离心机中短暂旋转管子。
将管子存放在零下80摄氏度，直到RNA提取完成。笔载玻片上被血液染色的肝脏切片在激光捕捉显微解剖前后均被显微镜观察。表现出微囊泡脂肪变的兴趣区域被精确收集到A型PCR管的盖子中。
保持细胞形态的完整。从约1000个和2000个激光捕获微切割分离细胞中提取的核糖核酸总量分别为110.7纳克和213.3纳克。所有样品均显示高纯度，吸收率介于1.9至2.1纳米之间，吸收率为260×280纳米。
凝胶电泳显示了明显的28s和18s核糖体核酸带。生物分析仪从少至1000个细胞中提取核糖核酸的谱。核糖核酸完整性数为6.7。
家务基因β肌动蛋白的Q-R-T-P-C-R得出的周期阈值范围为17至19。对由50纳克总核糖核酸构建的测序文库进行生物分析仪评估显示，其特征峰值在300至500碱基对之间。对这些文库进行RNA测序，数据质量高，Q20和Q30得分均超过90%，且重复间错误率较低。
一个重要的考虑是通过防腐处理、快速染色和加工过程中的直线条件来防止抗降解。按顺序操作，可用于实时PCR和带有碎片优化文库密钥的测序。肝脏样本中确立了这一点。
该方案支持肝细胞亚群的分析，并支持以病理为重点的肝病学研究。
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Overview
This article presents a detailed protocol for laser capture microdissection (LCM) and RNA extraction from paraformaldehyde (PFA)-fixed, OCT-embedded mouse liver sections. The method enables the isolation of specific histological regions for downstream transcriptomic analyses, ensuring high RNA purity and integrity suitable for sensitive applications such as qPCR and RNA sequencing.
Key Study Components
Area of Science
- Hepatology
- Molecular Biology
- Histology
Background
- Chronic liver diseases often require spatially resolved molecular analysis of hepatocyte subpopulations.
- Traditional FFPE methods can compromise RNA integrity, limiting downstream applications.
- LCM allows precise isolation of defined cell populations from complex tissues.
- PFA fixation and OCT embedding preserve tissue morphology and RNA quality for transcriptomic studies.
Purpose of Study
- To establish a robust protocol for isolating specific liver cell populations using LCM from PFA-fixed, OCT-embedded tissue.
- To extract high-quality RNA suitable for transcriptomic analysis from microscale samples.
- To validate the protocol's effectiveness for downstream applications such as qPCR and RNA sequencing.
Methods Used
- Preparation of pen membrane slides with UV exposure and poly-L-lysine coating.
- Cryosectioning of PFA-fixed, OCT-embedded mouse liver at -20°C.
- Hematoxylin staining and RNase-free handling throughout the workflow.
- Laser capture microdissection of histologically defined regions (approx. 1,000 cells).
- RNA extraction, quantification, and quality assessment (A260/A280 ratio, RIN, gel electrophoresis).
- Downstream validation by qPCR for β-actin and RNA sequencing with fragmentation-optimized library preparation.
Main Results
- RNA yields from 1,000–2,000 cells ranged from 110.7 to 213.3 ng.
- RNA purity was high (A260/A280: 1.9–2.1).
- RNA integrity number (RIN) averaged 6.7 ± 0.9, consistent with PFA fixation.
- qPCR for β-actin showed Ct values of 17–19, indicating good RNA quality.
- RNA-seq libraries showed characteristic peaks (300–500 bp) and >90% of bases at Q20/Q30 quality thresholds.
Conclusions
- This protocol enables precise, spatially resolved gene expression analysis in liver tissue.
- RNA extracted is of sufficient purity and integrity for sensitive downstream applications.
- The method supports hepatocyte subgroup profiling and pathology-focused hepatology research.
Frequently Asked Questions
Executive Industry Relevance
Laser capture microdissection (LCM) of paraformaldehyde-fixed, OCT-embedded mouse liver tissue enables spatially resolved transcriptomic profiling of defined cell populations, supporting mechanistic de-risking in early discovery. This approach provides high-purity RNA suitable for sensitive downstream analyses, facilitating confident target validation and disease-relevant pathway interrogation. The protocol addresses a critical inflection point for hepatology and pathology-focused R&D portfolios seeking robust, region-specific molecular insights.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables spatially precise interrogation of hepatocyte subgroups within complex liver tissue.
- Supports functional target validation by isolating histologically defined regions for RNA analysis.
- Facilitates mechanistic de-risking by linking molecular signatures to specific pathological features.
- Improves predictive confidence for portfolio triage by providing high-quality, region-specific RNA data.
Screening & Assay Development
- Prepares validated biological samples for downstream qPCR and RNA sequencing workflows.
- Ensures assay reproducibility through standardized tissue processing and RNA quality metrics.
- Delivers quantitative outputs (e.g., Ct values, RNA integrity) for reliable compound evaluation.
- Enables platform reuse for multiple transcriptomic applications in liver disease models.
Translational & Preclinical Research
- Aligns molecular profiling with disease-relevant histopathology for translational biomarker discovery.
- Maintains continuity from discovery through preclinical validation by supporting sensitive RNA-based analyses.
- Reduces biological risk in preclinical advancement decisions by ensuring sample integrity and data quality.
- Supports pathology-driven research in chronic liver disease models.
Pipeline & Workflow Integration
This LCM protocol integrates into the discovery-to-preclinical continuum by enabling targeted gene expression analysis from fixed tissue sections, bridging histological assessment and molecular readouts.
- Discovery Biology: Provides spatially resolved hypothesis testing and pathway clarification in liver tissue.
- Screening: Delivers reproducible, quantitative RNA outputs for assay development and validation.
- Analytics: Supplies high-purity RNA and robust quality metrics (A260/A280, RIN, Ct values) for comparative analyses.
- Translational Research: Connects histopathological features to transcriptomic profiles for biomarker alignment.
- Enterprise Reuse: Establishes a reusable workflow for spatial transcriptomics in fixed tissue models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in target validation.
- Operational Value: Standardizes sample preparation and RNA extraction for reproducibility and scalability.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient portfolio management.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of liver disease programs.
Implementation Considerations
- Requires expertise in cryosectioning, histological staining, and LCM instrumentation.
- Demands access to RNase-free facilities and analytical infrastructure for RNA quality assessment.
- Necessitates cross-team standardization of tissue processing and RNA extraction protocols.
- May require adaptation for other tissue types or disease models with validation.
- RNA integrity is inherently limited by PFA fixation but remains suitable for qPCR and RNA-seq.
