Morris Renée, Mehta Prachi
Translational Neuroscience Facility, School of Medical Sciences, The University of New South Wales (UNSW Sydney), Sydney, NSW, Australia.
Neurobiology Research Group, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia.
Methods Mol Biol. 2018;1723:223-233. doi: 10.1007/978-1-4939-7558-7_12.
In mammals, the central nervous system (CNS) is constituted of various cellular elements, posing a challenge to isolating specific cell types to investigate their expression profile. As a result, tissue homogenization is not amenable to analyses of motor neurons profiling as these represent less than 10% of the total spinal cord cell population. One way to tackle the problem of tissue heterogeneity and obtain meaningful genomic, proteomic, and transcriptomic profiling is to use laser capture microdissection technology (LCM). In this chapter, we describe protocols for the capture of isolated populations of motor neurons from spinal cord tissue sections and for downstream transcriptomic analysis of motor neurons with RT-PCR. We have also included a protocol for the immunological confirmation that the captured neurons are indeed motor neurons. Although focused on spinal cord motor neurons, these protocols can be easily optimized for the isolation of any CNS neurons.
在哺乳动物中，中枢神经系统（CNS）由各种细胞成分构成，这给分离特定细胞类型以研究其表达谱带来了挑战。因此，组织匀浆法不适用于运动神经元谱分析，因为运动神经元在脊髓细胞总数中所占比例不到10%。解决组织异质性问题并获得有意义的基因组、蛋白质组和转录组图谱的一种方法是使用激光捕获显微切割技术（LCM）。在本章中，我们描述了从脊髓组织切片中捕获分离的运动神经元群体的方案，以及使用逆转录聚合酶链反应（RT-PCR）对运动神经元进行下游转录组分析的方案。我们还纳入了一个用于免疫确认所捕获的神经元确实是运动神经元的方案。尽管这些方案侧重于脊髓运动神经元，但可以很容易地对其进行优化，以分离任何中枢神经系统神经元。
