Multiplexed, targeted gene expression profiling and genetic analysis on electronic microarrays.
BACKGROUND
Electronic microarrays comprise independent microelectrode test sites that can be electronically biased positive or negative, or left neutral, to move and concentrate charged molecules such as DNA and RNA to one or more test sites. We developed a protocol for multiplexed gene expression profiling of mRNA targets that uses electronic field-facilitated hybridization on electronic microarrays.


METHODS
A multiplexed, T7 RNA polymerase-mediated amplification method was used for expression profiling of target mRNAs from total cellular RNA; targets were detected by hybridization to sequence-specific capture oligonucleotides on electronic microarrays. Activation of individual test sites on the electronic microarray was used to target hybridization to designated subsets of sites and allow comparisons of target concentrations in different samples. We used multiplexed amplification and electronic field-facilitated hybridization to analyze expression of a model set of 10 target genes in the U937 cell line during lipopolysaccharide-mediated differentiation. Performance of multiple genetic analyses (single-nucleotide polymorphism detection, gene expression profiling, and splicing isoform detection) on a single electronic microarray was demonstrated using the ApoE and ApoER2 genes as a model system.


RESULTS
Targets were detected after a 2-min hybridization reaction. With noncomplementary capture probes, no signal was detectable. Twofold changes in target concentration were detectable throughout the ( approximately 64-fold) range of concentrations tested. Levels of 10 targets were analyzed side by side across seven time points. By confining electronic activation to subsets of test sites, polymorphism detection, expression profiling, and splicing isoform analysis were performed on a single electronic microarray.


CONCLUSIONS
Microelectronic array technology provides specific target detection and quantification with advantages over currently available methodologies for targeted gene expression profiling and combinatorial genomics testing.
Weidenhammer
Elaine M
EM
Department of Research and Development, Nanogen, Inc., 10398 Pacific Center Ct., San Diego, CA 92121, USA. eweidenhammer@nanogen.com
Kahl
Brenda F
BF
Wang
Ling
L
Wang
Larry
L
Duhon
Melanie
M
Jackson
Jo Ann
JA
Slater
Matthew
M
Xu
Xiao
X
D017398:Alternative Splicing; D001057:Apolipoproteins E / Q000235:genetics / Q000378:metabolism; D017123:Bacteriophage T7; D001921:Brain / Q000378:metabolism; D002454:Cell Differentiation; D012321:DNA-Directed RNA Polymerases; D004581:Electronics; D020869:Gene Expression Profiling; D006801:Humans; D026502:LDL-Receptor Related Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D018110:Receptors, Lipoprotein / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D020298:U937 Cells; D014764:Viral Proteins
D001057:Apolipoproteins E; D026502:LDL-Receptor Related Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D018110:Receptors, Lipoprotein; D014764:Viral Proteins; C098773:low density lipoprotein receptor-related protein 8; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases