在尸检人类脑组织上进行区域特异性原位杂交引导的激光捕获显微切割并结合基因表达定量分析。
Region-specific in situ hybridization-guided laser-capture microdissection on postmortem human brain tissue coupled with gene expression quantification.
作者信息
Bernard René, Burke Sharon, Kerman Ilan A
机构信息
Centrum für Anatomie, Institut für Integrative Neuroanatomie, Charité Campus Mitte-Universitätsmedizin Berlin, Berlin, Germany.
出版信息
Methods Mol Biol. 2011;755:345-61. doi: 10.1007/978-1-61779-163-5_29.
Abstract
This chapter describes the procedure of in situ hybridization-guided laser-capture microdissection performed on postmortem human brain tissue. This procedure permits the precise collection of brain tissue within anatomically defined brain nuclei that is enriched with mRNA. The chapter emphasizes the specific handling of postmortem tissue and preservation of RNA integrity to ensure high-quality gene profiling. Downstream procedures including mRNA amplification, gene profiling using high-density microarray chips, and confirmation with quantitative real-time polymerase chain reaction (qPCR) are described. PCR primer design and cDNA quantification required for qPCR are delineated.
摘要
本章描述了对死后人类脑组织进行原位杂交引导的激光捕获显微切割的程序。该程序允许在富含mRNA的解剖学定义的脑核内精确收集脑组织。本章强调了对死后组织的特殊处理以及RNA完整性的保存，以确保高质量的基因分析。还描述了包括mRNA扩增、使用高密度微阵列芯片进行基因分析以及通过定量实时聚合酶链反应（qPCR）进行确认等下游程序。阐述了qPCR所需的PCR引物设计和cDNA定量。
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