DNA self-polymers as microarray probes improve assay sensitivity.
DNA microarrays provide a method for determining the expression levels of thousands of genes simultaneously. However, the phenotypic complexity of brain tissue and cross-dilution of transcripts from different sources make it difficult to detect many of the low abundance RNA species. Furthermore, these experiments require significant amounts of starting material, which must often be amplified by one or two rounds of T7 amplification. We have developed a novel microarray probe with increased sensitivity. In this approach, PCR-generated microarray probes are end-ligated into redundant polymers and printed on standard arraying surfaces. These DNA polymer probes result in greatly improved sensitivity over classical monomer probes. Furthermore, polymer microarray sensitivity can be even further improved by incorporation of a biotin adapter into the first strand cDNA during reverse transcription and attachment of a gold particle (Genicon RLS, Invitrogen, CA) in a secondary reaction. This approach allowed us to reliably assess: expression of genes from < 5 microg of total RNA starting material without sample amplification. Finally, the resonance light scattering-labeled microarrays can be archived without fading, allowing re-scanning at a later time.
Hollingshead
Deborah
D
W941 Biomedical Science Tower, Genomics and Proteomics Core Laboratories, University of Pittsburgh, Pittsburgh, PA 15261, USA. hollings@pitt.edu
Korade
Zeljka
Z
Lewis
David A
DA
Levitt
Pat
P
Mirnics
Károly
K
D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics*; D019281:Dimerization; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004247:DNA