PDF(Pubmed)
Abstract：
The rat supraoptic nucleus (SON) contains magnocellular neurons that project long axons that terminate in the posterior pituitary gland. To perform molecular characterization of these regions, such as transcriptome and methylome profiling, it is necessary to obtain large quantities of high-quality RNA and DNA. Prior methods to isolate molecular material from these small regions required fixing or freezing and laser microdissection of whole tissue, which can compromise recovery and integrity. We have established a straight-forward method of dissecting out the SON and posterior pituitary gland from fresh, unfixed tissue that allows for the isolation of RNA or DNA without compromising nucleic acid integrity. Furthermore, this method can be used as a framework for the microdissection of any region of the brain to isolate any sensitive material. In this manuscript, we describe step-by-step instructions from the macro scale dissection, to brain sectioning, and finally the microdissection of the appropriate tissue.•Transcardial perfusion without fixative prevents the shortcomings of nucleic acid cross-linking.•A fast method and the maintenance of tissue in ice-cold HBSS during dissection and sectioning prevents nucleic acid degradation.•A vibratome is used for the sectioning of fresh brain tissue without freezing or gelatin embedding (i.e. cryostat or microtome).
摘要：
大鼠视上核（SON）包含大细胞神经元，这些神经元投射终止于垂体后叶的长轴突。为了对这些区域进行分子表征，例如转录组和甲基化组分析，有必要获得大量高质量的RNA和DNA。从这些小区域分离分子材料的现有方法需要固定或冷冻和激光显微切割整个组织，这可能会损害恢复和完整性。我们已经建立了一种直接的方法，从新鲜的，允许在不损害核酸完整性的情况下分离RNA或DNA的未固定组织。此外，这种方法可以用作显微解剖大脑任何区域的框架，以分离任何敏感物质。在这份手稿中,我们描述了从宏观尺度解剖的逐步指令，大脑切片，最后是适当组织的显微解剖。•没有固定剂的经心脏灌注防止核酸交联的缺点。•快速方法和在解剖和切片期间在冰冷的HBSS中维持组织防止核酸降解。•振动切片机用于在没有冷冻或明胶包埋的情况下切片新鲜的脑组织(即低温恒温器或切片机)。
