Nicot Arnaud B, Rambeau Justine, Guillot Flora, Garcia Alexandra, Laplaud David A
Université de Nantes, Université Bretagne Loire (UBL), Nantes, France.
Institut de Transplantation Urologie Néphrologie (ITUN)/CESTI, CHU Nantes, Nantes, France.
Methods Mol Biol. 2018;1723:261-271. doi: 10.1007/978-1-4939-7558-7_15.
Laser-capture microdissection (LCM) allows for retrieval of specific cell populations in situ. By combining immunofluorescent labeling with LCM, mRNAs can be probed by qRT-PCR for determining in situ gene expression during health and disease. This approach permits obtaining and analyzing histologically enriched cell populations in a tissue that can be hardly obtained from other methods such as white matter astrocytes from rodents or any individual cell population from archival human or rodent brain tissues. Herein, we present our methodology of laser-captured mouse spinal cord white matter astrocytes, which can be adapted for any cell type in CNS tissue and low RNAse containing tissues. The methods presented with an emphasis on tips and advices include the cryostat section preparation from snap-frozen tissue, an adapted immunofluorescent labeling, a brief overview of LCM using a UV-based technology with polyethylene membrane glass slides, procedures for direct use of RNA from lysis buffer vs. column-based purified RNA, RNA quality/quantity assessment, the reverse transcription and preamplification steps used before real-time qPCR analysis.
激光捕获显微切割（LCM）能够在原位获取特定的细胞群体。通过将免疫荧光标记与LCM相结合，可利用qRT-PCR检测mRNA，以确定健康和疾病状态下的原位基因表达。这种方法能够获取并分析组织中组织学上富集的细胞群体，而这些细胞群体很难通过其他方法获得，例如来自啮齿动物的白质星形胶质细胞，或来自存档的人类或啮齿动物脑组织的任何单个细胞群体。在此，我们展示了激光捕获小鼠脊髓白质星形胶质细胞的方法，该方法可适用于中枢神经系统组织和低核糖核酸酶组织中的任何细胞类型。本文介绍的方法重点在于技巧和建议，包括从速冻组织制备冰冻切片、改良的免疫荧光标记、使用基于紫外线技术和聚乙烯膜载玻片的LCM简要概述、直接使用裂解缓冲液中的RNA与基于柱纯化RNA的步骤、RNA质量/数量评估、实时定量PCR分析前的逆转录和预扩增步骤。
