DNA self-polymers as microarray probes improve assay sensitivity
Descrição do Produto
Journal of Neuroscience Methods 151 (2006) 216–223
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DNA self-polymers as microarray probes improve assay sensitivity ˇ Deborah Hollingshead a,∗∗ , Zeljka Korade b,1 , David A. Lewis c,d,2 , Pat Levitt e,3 , K´aroly Mirnics b,f,∗ b
a W941 Biomedical Science Tower, Genomics and Proteomics Core Laboratories, University of Pittsburgh, Pittsburgh, PA 15261, USA Pediatric Center for Neuroscience, Department of Pediatrics, 7116 Rangos Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA c W1650 Biomedical Science Tower, Department of Psychiatry, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261, USA d Department Neuroscience, University of Pittsburgh, Pittsburgh, PA 15261, USA e Kennedy Center for Research on Human Development, Vanderbilt University, P.O. Box 40 Peabody, Nashville, TN 37203, USA f Department of Neurobiology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261, USA
Received 20 May 2005; received in revised form 18 July 2005; accepted 20 July 2005
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