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Nucleic Acids Research 2004 32(16):e126; doi:10.1093/nar/gnh119
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Published online 8 September 2004

Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved

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*

ACLARA BioSciences, Inc., 1288 Pear Avenue, Mountain View, CA 94043, USA

* To whom correspondence should be addressed. Tel: +1 650 210 1247; Fax: +1 650 210 1210; Email: ssingh{at}aclara.com
Correspondence may also be addressed to Huan Tian. Tel: +1 650 210 1289; Fax: +1 650 210 1210; Email: ttian{at}aclara.com

Received November 7, 2003; Revised April 23, 2004; Accepted August 8, 2004

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 1ß. 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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