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

Nucleic Acids Research
Huan TianSharat Singh

Abstract

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 ...Continue Reading

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Citations

Jul 17, 2008·Comparative and Functional Genomics·J S AlmeidaG Warr
Jun 14, 2005·Expert Opinion on Therapeutic Targets·Ruo-Pan HuangRuochun Huang
Jun 3, 2006·Journal of Microbiological Methods·Kari KatajaHans Söderlund
Mar 21, 2006·Analytical Biochemistry·Michael FlagellaYuling Luo
Jun 24, 2008·Journal of Biomolecular Screening·Xiaohua Douglas Zhang

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