Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis
Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis
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DOI：
10.1038/nprot.2007.79
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Saitou, Mitinori
中科院分区：
文献类型：
--
作者：
Kurimoto, Kazuki;Yabuta, Yukihiro;Saitou, Mitinori
我们在这里描述了从典型的单个哺乳动物细胞中代表性扩增全局mRNA的方案，以提供高密度寡核苷酸微阵列分析的模板。单个细胞在没有纯化的管子中裂解，并使用聚（DT）尾底漆合成第一链cDNA。未反应的底漆是通过外核酸酶处理特异性消除的，第二链是在第一链cDNA的聚（DA）尾巴后用第二个聚（DT）尾底漆产生的。 cDNA分为四个管，通过PCR独立地定向，然后重新组合。放大产物（类似于100 ng）显示出原始基因表达的较高表示和可重复性，尤其是针对每个细胞的20份副本表达的基因，与通过常规PCR协议获得的基因相比，可以有效地用于定量PCR和定量PCR和EST分析。然后将cDNA与带有T7启动子序列的引物进行另一个PCR扩增。所得的cDNA产物凝胶纯化，通过一个最终周期扩增，用于通过T7 RNA聚合酶进行等温线性扩增，以合成CRNA，以进行微阵列杂交。该协议产生的cDNA模板足以实现单个单元的80多个微阵列杂交，并且可以在5-6天内完成。
We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly( dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (similar to 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.