Bernard René, Kerman Ilan A, Meng Fan, Evans Simon J, Amrein Irmgard, Jones Edward G, Bunney William E, Akil Huda, Watson Stanley J, Thompson Robert C
Molecular & Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA.
J Neurosci Methods. 2009 Mar 30;178(1):46-54. doi: 10.1016/j.jneumeth.2008.11.012. Epub 2008 Nov 25.
Laser capture microdissection (LCM) permits isolation of specific cell types and cell groups based upon morphology, anatomical landmarks and histochemical properties. This powerful technique can be used for region-specific dissection if the target structure is clearly delineated. However, it is difficult to visualize anatomical boundaries in an unstained specimen, while histological staining can complicate the microdissection process and compromise downstream processing and analysis. We now introduce a novel method in which in situ hybridization (ISH) signal is used to guide LCM on adjacent unstained sections to collect tissue from neurochemically defined regions of the human postmortem brain to minimize sample manipulation prior to analysis. This approach was validated in nuclei that provide monoaminergic inputs to the forebrain, and likely contribute to the pathophysiology of mood disorders. This method was used successfully to carry out gene expression profiling and quantitative real-time PCR (qPCR) confirmation from the dissected material. When compared to traditional micropunch dissections, our ISH-guided LCM method provided enhanced signal intensity for mRNAs of specific monoaminergic marker genes as measured by genome-wide gene expression microarrays. Enriched expression of specific monoaminergic genes (as determined by microarrays and qPCR) was detected within appropriate anatomical locations validating the accuracy of microdissection. Together these results support the conclusion that ISH-guided LCM permits acquisition of enriched nucleus-specific RNA that can be successfully used for downstream gene expression investigations. Future studies will utilize this approach for gene expression profiling of neurochemically defined regions of postmortem brains collected from mood disorder patients.
激光捕获显微切割（LCM）技术可根据细胞形态、解剖标志和组织化学特性分离特定的细胞类型和细胞群。如果目标结构清晰可辨，这项强大的技术可用于区域特异性切割。然而，在未染色的标本中很难看清解剖边界，而组织学染色会使显微切割过程变得复杂，并影响后续的处理和分析。我们现在介绍一种新方法，即利用原位杂交（ISH）信号在相邻的未染色切片上指导LCM，从人类死后大脑的神经化学定义区域收集组织，以尽量减少分析前的样本操作。该方法在前脑单胺能输入核中得到验证，这些核可能与情绪障碍的病理生理学有关。该方法成功用于从切割材料中进行基因表达谱分析和定量实时PCR（qPCR）验证。与传统的微量打孔切割相比，我们的ISH引导LCM方法通过全基因组基因表达微阵列检测，为特定单胺能标记基因的mRNA提供了更强的信号强度。在适当的解剖位置检测到特定单胺能基因的富集表达（通过微阵列和qPCR确定），验证了显微切割的准确性。这些结果共同支持了这样的结论，即ISH引导的LCM能够获取富集的核特异性RNA，可成功用于下游基因表达研究。未来的研究将利用这种方法对从情绪障碍患者收集的死后大脑神经化学定义区域进行基因表达谱分析。
