title: Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.
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 assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.
authors.affiliation: ACLARA BioSciences, Inc., 1288 Pear Avenue, Mountain View, CA 94043, USA. ttian@aclara.com
authors.forename: Huan
authors.initials: H
authors.lastname: Tian
authors.forename: Liching
authors.initials: L
authors.lastname: Cao
authors.forename: Yuping
authors.initials: Y
authors.lastname: Tan
authors.forename: Stephen
authors.initials: S
authors.lastname: Williams
authors.forename: Lili
authors.initials: L
authors.lastname: Chen
authors.forename: Tracy
authors.initials: T
authors.lastname: Matray
authors.forename: Ahmed
authors.initials: A
authors.lastname: Chenna
authors.forename: Sean
authors.initials: S
authors.lastname: Moore
authors.forename: Vincent
authors.initials: V
authors.lastname: Hernandez
authors.forename: Vivian
authors.initials: V
authors.lastname: Xiao
authors.forename: Mengxiang
authors.initials: M
authors.lastname: Tang
authors.forename: Sharat
authors.initials: S
authors.lastname: Singh
mesh_terms: D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007700:Kinetics; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger