Martuscello Regina T, Louis Elan D, Faust Phyllis L
Department of Pathology and Cell Biology, Columbia University.
Division of Movement Disorders, Department of Neurology, Yale University; Department of Chronic Disease Epidemiology, Yale School of Public Health, Yale University; Center for Neuroepidemiology and Clinical Neurological Research, Yale School of Medicine, Yale University.
J Vis Exp. 2019 Jan 17(143). doi: 10.3791/58953.
Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (≥8) as needed for transcriptional profiling experiments.
激光捕获显微切割（LCM）是一种有利的工具，可用于从异质性组织中收集细胞学和/或表型相关的细胞或区域。捕获的产物可用于多种分子方法以分离蛋白质、DNA或RNA。然而，从死后人类脑组织中保存RNA尤其具有挑战性。LCM的标准可视化技术需要组织学或免疫组织化学染色程序，这可能会进一步降解RNA。因此，我们设计了一种用于LCM可视化的无染色方案，目的是在死后人类脑组织中保存RNA完整性。小脑的浦肯野细胞由于其大小和特征性位置，是无染色可视化的良好候选对象。小脑皮质有不同的层，细胞密度不同，使其成为在高倍显微镜下识别的良好原型。浦肯野细胞是位于颗粒细胞层（由小神经元组成的密集细胞网络）和分子层（细胞体稀疏）之间的大神经元。由于这种结构，使用无染色可视化是可行的。其他模仿这种表型的器官或细胞系统也将是合适的。无染色方案旨在用乙醇固定新鲜冷冻组织，并用二甲苯去除脂质，以在高倍光学显微镜下改善形态学可视化。该方案不考虑其他固定方法，专门为使用紫外（UV）-LCM系统捕获的新鲜冷冻组织样本设计。在这里，我们展示了一个完整的方案，用于对新鲜冷冻的死后人类小脑组织进行切片和固定，以及从通过UV-LCM分离的浦肯野细胞中纯化RNA，同时保留RNA质量以用于后续的RNA测序。在我们的操作中，该方案无需染色试剂就能产生出色的细胞可视化水平，并能产生转录谱实验所需的具有高RNA完整性数值（≥8）的RNA。
