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使用cDNA微阵列进行基因表达分析。

Gene expression analysis using cDNA microarrays.

作者信息

Karsten Stanislav L, Geshwind Daniel H

机构信息

UCLA School of Medicine, Los Angeles, California, USA.

出版信息

Curr Protoc Neurosci. 2002 Nov;Chapter 4:Unit 4.28. doi: 10.1002/0471142301.ns0428s20.

Abstract

This unit focuses on cDNA microarrays, which are being increasingly applied to large-scale gene expression studies in the nervous system, concentrating on the steps of probe preparation and microarray hybridization. Four protocols for the attachment of fluorophores to cDNA (labeling) and cDNA hybridization onto microarrays are presented: direct labeling using either Klenow fragment or reverse transcriptase, and two signal amplification techniques represented by the tyramide signal amplification method (TSA), and detection and labeling of cDNA using PCR amplification. Basic issues in experimental design, sources of variability and potential pitfalls in microarray experiments are described. Anticipated results and follow up of microarray experiments, including confirmation of the results and troubleshooting are also discussed.

摘要

本单元聚焦于cDNA微阵列,其在神经系统大规模基因表达研究中的应用日益广泛,重点关注探针制备和微阵列杂交步骤。文中介绍了四种将荧光团连接到cDNA(标记)以及cDNA与微阵列杂交的方法:使用klenow片段或逆转录酶进行直接标记,以及以酪胺信号放大法(TSA)为代表的两种信号放大技术,还有利用PCR扩增对cDNA进行检测和标记。文中描述了实验设计中的基本问题、微阵列实验中变异性的来源及潜在陷阱。还讨论了微阵列实验的预期结果及后续工作,包括结果的确认和故障排除。

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