Byrne M C, Whitley M Z, Follettie M T
Genetics Institute/Wyeth Research, Cambridge, Massachusetts, USA.
Curr Protoc Neurosci. 2001 Nov;Chapter 4:Unit 4.26. doi: 10.1002/0471142301.ns0426s16.
The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A(+)) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.
构建包含数百至数千个基因的综合基因表达谱的能力,这些基因的RNA水平可同时进行监测,这代表了分子生物学中一项令人兴奋的新能力。这是通过将已定量扩增并用生物素标记的mRNA与DNA芯片杂交来实现的,该芯片展示了数千个与感兴趣的mRNA互补的核苷酸。在本单元中,讨论了从聚腺苷酸(+)RNA与总RNA开始的基本原理,并介绍了选择用于芯片设计的寡核苷酸的策略。还包括RNA扩增和标记、cDNA及体外转录产物的纯化和定量的方案。