DNA self-polymers as microarray probes improve assay sensitivity

Deborah Hollingshead, Željka Korade, David A. Lewis, Pat Levitt, Károly Mirnics

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

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 μg 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.

Original languageEnglish (US)
Pages (from-to)216-223
Number of pages8
JournalJournal of Neuroscience Methods
Volume151
Issue number2
DOIs
StatePublished - Mar 15 2006

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Keywords

  • DNA microarray
  • DNA polymerisation
  • Hybridization
  • Low abundance transcripts
  • Resonance light scattering
  • Self-ligation
  • Transcriptome profiling

ASJC Scopus subject areas

  • Neuroscience(all)

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