Assessment of differential gene expression in vestibular epithelial cell types using microarray analysis
Assessment of differential gene expression in vestibular epithelial cell types using microarray analysis
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DOI：
10.1016/j.molbrainres.2004.10.001
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发表时间：
2005-01-05
期刊：
影响因子：
--
通讯作者：
Popper, P
中科院分区：
文献类型：
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作者：
Cristobal, R;Wackym, PA;Popper, P
目前的全球基因表达技术允许在一个实验中评估和比较数千个基因的表达，提供了大量的信息。然而，这些技术产生的数据是上下文相关的，个别生物样本、RNA获取、扩增、杂交协议和基因芯片制备方法以及硬件和分析软件的微小差异导致结果之间的相关性较差。目前面临的重大困难之一是使议定书标准化，以便对结果进行有意义的比较。在内耳，由于不同细胞类型的高密度和组织的稀少，从单个细胞群中获取RNA仍然是一个挑战。因此，激光捕获显微切割被用来选择性地从壶腹隆起收集单个细胞和细胞区域，然后提取总RNA并扩增到足以进行高通量分析的数量。用逆转录聚合酶链式反应(RT-PCR)扩增毛细胞特异性基因表达谱，扩增出肌球蛋白VIIA、钙调蛋白和α9烟碱型乙酰胆碱受体亚单位mRNAs。为了证实支持细胞特异性基因的表达，用RT-PCR方法扩增了细胞周期蛋白依赖性激酶抑制因子p27kip1的mRNA。随后的A9 RT-PCR实验证明了I型毛细胞和11型毛细胞之间的表型差异，只在11型毛细胞中表达。利用激光捕获显微切割技术，微阵列表达谱显示408个基因在毛细胞和支持细胞之间的表达差异超过5倍，其中175个得到了很好的注释。有97个注释基因的毛细胞相对于支持细胞的表达差异大于5倍，78个注释基因的支持细胞相对于毛细胞的表达差异大于5倍。(C)2004爱思唯尔B.V.保留所有权利。
Current global gene expression techniques allow the evaluation and comparison of the expression of thousands of genes in a single experiment, providing a tremendous amount of information. However, the data generated by these techniques are context-dependent, and minor differences in the individual biological samples, methodologies for RNA acquisition, amplification, hybridization protocol and gene chip preparation, as well as hardware and analysis software, lead to poor correlation between the results. One of the significant difficulties presently faced is the standardization of the protocols for the meaningful comparison of results. In the inner ear, the acquisition of RNA from individual cell populations remains a challenge due to the high density of the different cell types and the paucity of tissue. Consequently, laser capture microdissection was used to selectively collect individual cells and regions of cells from cristae ampullares followed by extraction of total RNA and amplification to amounts sufficient for high throughput analysis. To demonstrate hair cell-specific gene expression, myosin VIIA, calmodulin and (alpha9 nicotinic acetylcholine receptor subunit mRNAs were amplified using reverse transcription-polymerase chain reaction (RT-PCR). To demonstrate supporting cell-specific gene expression, cyclin-dependent kinase inhibitor p27kip1 mRNA was amplified using RT-PCR. Subsequent experiments with a9 RT-PCR demonstrated phenotypic differences between type I and type 11 hair cells, with expression only in type 11 hair cells. Using the laser capture microdissection technique, microarray expression profiling demonstrated 408 genes with more than a five-fold difference in expression between the hair cells and supporting cells, of these 175 were well annotated. There were 97 annotated genes with greater than a five-fold expression difference in the hair cells relative to the supporting cells, and 78 annotated genes with greater than a five-fold expression difference in the supporting cells relative to the hair cells. (C) 2004 Elsevier B.V. All rights reserved.