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首页> 外文期刊>Brain research. Brain research protocols >Gene expression profiles derived from single cells in human postmortem brain.
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Gene expression profiles derived from single cells in human postmortem brain.

机译:基因表达谱来自人死后大脑中的单个细胞。

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摘要

The study of postmortem human brain tissue remains the basis for the understanding of many CNS disorders and to verify data obtained in experimental studies. So far, however, gene expression profiling in cellular sub-populations derived from human postmortem brain was hampered by several technical drawbacks. Here, we describe a method that allows the generation of mRNA expression profiles from single neurons. Dopaminergic neurons from different midbrain areas including substantia nigra, central gray substance and ventral tegmental area were identified and isolated by immuno-laser capture microscopy (LCM). Expression profiles were generated from microdissected cells using a modified RNA fingerprinting protocol. Using this approach, we were able to generate specific RNA fingerprints at a high resolution from phenotype-specific single neurons. Polymorphic fragments were isolated from gels and differential gene expression was confirmed by real-time PCR using gene-specific primer pairs and hybridization probes. The method described here is easy to use and reliable for profiling gene expression at the single cell level in human postmortem brain. It could therefore be valuable to open new insights into the molecular pathogenesis of CNS disorders.
机译:死后人类大脑组织的研究仍然是了解许多中枢神经系统疾病和验证实验研究中获得的数据的基础。然而,到目前为止,人类死后大脑衍生的细胞亚群中的基因表达谱受到一些技术缺陷的阻碍。在这里,我们描述了一种允许从单个神经元生成mRNA表达谱的方法。通过免疫激光捕获显微镜(LCM)鉴定并分离了来自中脑不同区域(包括黑质,中央灰色物质和腹侧被盖区)的多巴胺能神经元。使用修饰的RNA指纹方案从显微解剖的细胞生成表达谱。使用这种方法,我们能够从表型特定的单个神经元高分辨率生成特定的RNA指纹。从凝胶分离多态性片段,并使用基因特异性引物对和杂交探针通过实时PCR确认差异基因表达。本文描述的方法易于使用,并且在人死后大脑中单细胞水平上分析基因表达的方法可靠。因此,对于中枢神经系统疾病的分子发病机理开辟新见解可能是有价值的。

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