Gene Expression Analysis of Neurons and Astrocytes Isolated by Laser Capture Microdissection from Frozen Human Brain Tissues.
Gene Expression Analysis of Neurons and Astrocytes Isolated by Laser Capture Microdissection from Frozen Human Brain Tissues.
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DOI：
10.3389/fnmol.2016.00072
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发表时间：
2016
影响因子：
4.8
通讯作者：
Chiba-Falek O
中科院分区：
文献类型：
--
作者：
Tagliafierro L;Bonawitz K;Glenn OC;Chiba-Falek O
不同的细胞类型和多种细胞连接是人脑的特征。使用特定细胞群的基因表达分析比进行整个组织匀浆的分析更准确，特别是在神经退行性疾病的情况下，其中特定的细胞亚群受到不同病理学的影响。由于获得同质细胞群的困难，特定细胞类型（神经元、星形胶质细胞等）中的基因表达不稳定。已经被低估了利用档案资源的冷冻人脑在神经退行性疾病的研究，我们开发和校准的方法来量化细胞类型特异性神经元，星形胶质细胞的表达谱的基因参与神经退行性疾病，包括帕金森氏症和阿尔茨海默氏症。使用存档的人冷冻脑组织制备载玻片，用于使用细胞特异性抗体进行快速免疫染色。激光捕获显微切割（LCM）分离免疫反应阳性细胞。在分析后阶段，通过细胞特异性标志物的表达来验证特定感兴趣细胞类型的富集。我们优化了该技术以保持RNA的完整性，使得RNA适合于下游表达分析。在RNA提取后，使用nCounter单细胞基因表达测定（NanoString Technologies®）以数字方式确定表达水平。结果表明，使用我们优化的技术，我们成功地从人冷冻脑组织中分离出单个神经元和星形胶质细胞，并获得了质量良好的RNA，适用于mRNA表达分析。与以前报道的方法相比，我们在这里提出了新的进展，这提高了该方法的可行性及其对各种下游分子分析的适用性。我们新开发的方法可以在神经退行性疾病的遗传和功能基因组研究中实施，并有可能显着推进该领域。
Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty of obtaining homogenous cell populations, gene expression in specific cell-types (neurons, astrocytes, etc.) has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific—neuronal, astrocytes—expression profiles of genes implicated in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM). The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies®). The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method's feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the potential to significantly advance the field.