Haupt Corinna, Tolner Else A, Heinemann Uwe, Witte Otto W, Frahm Christiane
Department of Neurology, Friedrich-Schiller-University, Jena, Germany.
Brain Res. 2006 Nov 6;1118(1):232-8. doi: 10.1016/j.brainres.2006.08.037. Epub 2006 Sep 20.
Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.
病理生理状态下的基因表达变化可通过原位杂交进行时空监测，并通过实时逆转录聚合酶链反应（RT-PCR）进行可靠定量。在此，我们开发了一种新方法，即冷冻切片的相邻载玻片可用于通过原位杂交和实时RT-PCR进行基因表达分析。我们应用该方法评估大鼠海马体中，在海藻酸诱导癫痫发作后，主要星形胶质细胞连接蛋白连接蛋白43（Cx43）的mRNA表达。形成间隙连接的连接蛋白在包括癫痫在内的几种脑部疾病的发病机制中起作用。通过对与地高辛标记的RNA探针杂交的脑切片进行计算机图像分析，自动定量海藻酸处理组和对照组大鼠海马体中Cx43 mRNA阳性细胞的数量。同时，使用实时RT-PCR检测相邻脑切片海马组织中相对的Cx43 mRNA水平。应用这两种非常灵敏的方法，我们发现海藻酸诱导的癫痫发作不影响海马体连接蛋白43 mRNA的表达。
