Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.
Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.
复制标题
使用电泳标签进行多重 mRNA 测定，进行高通量基因表达分析。
DOI：
10.1093/nar/gnh119
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发表时间：
2004
影响因子：
14.9
通讯作者：
Sharat Singh
中科院分区：
文献类型：
--
作者：
Huan Tian;L. Cao;Yuping Tan;Stephen J. Williams;Lili Chen;T. Matray;A. Chenna;S. Moore;Vincent Hernandez;Vivian Xiao;Mengxiang Tang;Sharat Singh
我们描述了一种新的多路复用技术，使用一个小的荧光分子，称为eTag分子库，编码和量化mRNA的目标。eTag分子具有相同的荧光特性，但具有不同的荷质比，具有预定义的电泳特性，可以使用毛细管电泳进行解析。结合初级Invader mRNA测定，应用eTag分子同时定量多达44种mRNA靶标。通过检查用炎性细胞因子白细胞介素1 β刺激的人脐静脉内皮细胞中的一组炎症反应基因，验证了这种多重方法。毛细管电泳中的激光诱导荧光检测和电动样品进样过程允许在反应中灵敏地定量数千个mRNA分子拷贝。该测定是精确的，如通过测量合格的Z'因子所评估的，Z'因子是用于使用mRNA测定的高通量筛选的无量纲和简单的特征。我们的数据表明，通过灵敏的毛细管电泳检测和等温线性扩增特性的入侵者测定的eTag分子的多路复用能力之间的协同作用。eTag多重mRNA检测为灵敏、高样品通量和多重基因表达分析提供了独特的平台。
We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1beta. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z' factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.
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影响因子：
--
作者：
V. Dolnik
通讯作者：
V. Dolnik
DOI：
10.1073/pnas.86.4.1173
发表时间：
1989-02-01
影响因子：
11.1
作者：
KWOH, DY;DAVIS, GR;GINGERAS, TR
通讯作者：
GINGERAS, TR
影响因子：
56.9
作者：
BEVILACQUA, MP;STENGELIN, S;SEED, B
通讯作者：
SEED, B
影响因子：
3.6
作者：
Thomas, RS;Rank, DR;Bradfield, CA
通讯作者：
Bradfield, CA
DOI：
10.1073/pnas.89.7.3010
发表时间：
1992-04-01
影响因子：
11.1
作者：
EBERWINE, J;YEH, H;COLEMAN, P
通讯作者：
COLEMAN, P