Obtaining high quality RNA from single cell populations in human postmortem brain tissue.
Obtaining high quality RNA from single cell populations in human postmortem brain tissue.
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DOI：
10.3791/1444
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发表时间：
2009-08
期刊：
影响因子：
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通讯作者：
C. Pietersen;M. P. Lim;T. Woo
中科院分区：
文献类型：
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作者：
C. Pietersen;M. P. Lim;T. Woo
我们建议通过询问第三层锥体神经元的基因表达谱来研究精神分裂症患者上级颞回的灰质减少。众所周知，大脑皮层是一种异常异质的结构，包括不同的区域、层和细胞类型，其中每一个的特征在于不同的细胞和分子组成，因此具有差异的基因表达谱。为了避免组织异质性的混杂效应，我们使用激光捕获显微切割（LCM）来分离我们的特定细胞类型，即锥体神经元。使用Arcturus XT LCM系统捕获约500个用Histogene染色溶液染色的锥体神经元。然后从捕获的细胞中分离RNA，并使用Arcturus/Molecular Devices试剂盒进行两轮基于T7的线性扩增。Experion LabChip（Bio-Rad）凝胶和电泳图表明a（m）RNA质量良好，转录物长度超过微阵列所需的600 nt。获得的mRNA的量平均为51微克，具有可接受的平均样品纯度，如由A260/280比率2.5所指示的。使用来自Affytechnology的人X3 P基因芯片探针阵列分析基因表达。
We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons. Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51 microg, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.