Garcia-Ovejero Daniel, Paniagua-Torija Beatriz, Arevalo-Martin Angel, Navarro-Galve Beatriz, Molina-Holgado Eduardo
Laboratory of Neuroinflammation, Hospital Nacional de Paraplejicos (SESCAM), Toledo, Spain.
Basic Biomedical Sciences Department, Health and Biomedical Sciences School, Universidad Europea de Madrid (UEM), Madrid, Spain.
Methods Mol Biol. 2018;1723:285-318. doi: 10.1007/978-1-4939-7558-7_17.
In the last few decades many efforts have been dedicated to decipher the nature and regenerative potential of neurogenic niches and endogenous stem cells after damage of the central nervous system. In the spinal cord, it has been largely focused on the ependymal region, which hosts neural precursors/stem cells (NSC) in rodents but differs between species and ages. In the current chapter, we detail our protocol to study the gene expression profile of this region using fresh frozen blocks of rat and human post-mortem spinal cords. We describe how to prepare and process those tissues, how to identify and dissect the ependymal region using Laser-Capture Microdissection (LCMD), and how to isolate and amplify RNA with different integrity states to finally obtain enough material for performing gene expression assays using Taqman Low Density Arrays. LCMD technique maintains tissue integrity allowing for subsequent analysis without manipulation steps that may alter molecular properties of cells and the eventual loss of delicate cell types in comparison with other approaches that require previous disaggregation of the tissue and cell manipulation before isolation.
在过去几十年中，人们付出了诸多努力来破译中枢神经系统受损后神经源性微环境和内源性干细胞的性质及再生潜力。在脊髓方面，主要聚焦于室管膜区域，该区域在啮齿动物中存在神经前体/干细胞（NSC），但不同物种和年龄之间存在差异。在本章中，我们详细介绍了使用大鼠和人类死后脊髓的新鲜冷冻切片来研究该区域基因表达谱的方案。我们描述了如何制备和处理这些组织，如何使用激光捕获显微切割（LCMD）来识别和解剖室管膜区域，以及如何分离和扩增具有不同完整性状态的RNA，最终获得足够的材料以使用Taqman低密度阵列进行基因表达分析。与其他需要在分离前对组织进行预先解离和细胞操作的方法相比，LCMD技术可保持组织完整性，从而允许后续分析，而无需可能改变细胞分子特性的操作步骤以及最终导致脆弱细胞类型丢失的步骤。
