Journal Article

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

Huan Tian, Liching Cao, Yuping Tan, Stephen Williams, Lili Chen, Tracy Matray, Ahmed Chenna, Sean Moore, Vincent Hernandez, Vivian Xiao, Mengxiang Tang and Sharat Singh

in Nucleic Acids Research

Volume 32, issue 16, pages e126-e126
Published in print September 2004 | ISSN: 0305-1048
Published online September 2004 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gnh119
Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

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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 1β. 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.

Journal Article.  6560 words.  Illustrated.

Subjects: Research Methods in Life Sciences

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