Modified serial analysis of gene expression method for construction of gene expression profiles of microbial eukaryotic species
Modified serial analysis of gene expression method for construction of gene expression profiles of microbial eukaryotic species
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DOI：
10.1128/aem.70.9.5298-5304.2004
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发表时间：
2004-09-01
影响因子：
4.4
通讯作者：
Cary, SC
中科院分区：
文献类型：
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作者：
Coyne, KJ;Burkholder, JM;Cary, SC
基因表达序列分析(SAGE)是鉴定差异表达基因的有效方法，它以短标签序列的形式提供了全面和定量的基因表达谱。每个标签代表一个独特的转录本，SAGE文库中标签的相对频率等于它们所代表的转录本的相对比例。从微生物制备SAGE文库的主要障碍之一是需要大量的起始材料(即信使核糖核酸)。在这里，我们提出了一种新的方法，通过使用Y连接子选择性地扩增3‘cDNA片段，从少量的总RNA中构建SAGE文库。为了验证这种方法，我们构建了有毒甲藻Pfiester shumway ae的全面基因表达谱。鼠尾草文库是由一种剧毒的鱼饵养殖的Shumwayae和一种最近有毒的藻类养殖而构建的。通过比较两个文库中的标签序列，鉴定了舒氏并殖吸虫特异的基因转录本。选择频率在库中标签总数的0.026到3.3%之间的代表性标签进行进一步分析。每一种转录物在单独的对照培养中都得到了证实。这里描述的改进的SAGE方法产生了似乎既全面又定量的基因表达谱，它直接适用于其他环境相关微生物物种的基因表达研究。
Serial analysis of gene expression (SAGE) is a powerful approach for the identification of differentially expressed genes, providing comprehensive and quantitative gene expression profiles in the form of short tag sequences. Each tag represents a unique transcript, and the relative frequencies of tags in the SAGE library are equal to the relative proportions of the transcripts they represent. One of the major obstacles in the preparation of SAGE libraries from microorganisms is the requirement for large amounts of starting material (i.e., mRNA). Here, we present a novel approach for the construction of SAGE libraries from small quantities of total RNA by using Y linkers to selectively amplify 3' cDNA fragments. To validate this method, we constructed comprehensive gene expression profiles of the toxic dinoflagellate Pfiesteria shumwayae. SAGE libraries were constructed from an actively toxic fish-fed culture of P. shumwayae and from a recently toxic alga-fed culture. P. shumwayae-specific gene transcripts were identified by comparison of tag sequences in the two libraries. Representative tags with frequencies ranging from 0.026 to 3.3% of the total number of tags in the libraries were chosen for further analysis. Expression of each transcript was confirmed in separate control cultures of toxic P. shumwayae. The modified SAGE method described here produces gene expression profiles that appear to be both comprehensive and quantitative, and it is directly applicable to the study of gene expression in other environmentally relevant microbial species.