Use of a three-color cDNA microarray platform to measure and control support-bound probe for improved data quality and reproducibility

Authors: Martin J. Hessner¹; Xujing Wang¹; Shehnaz Khan²; Lisa Meyer¹; Michael Schlicht³; Jennifer Tackes²; Milton W. Datta²; Howard J. Jacob²; Soumitra Ghosh¹

Source: Nucleic Acids Research, Volume 31, Number 11, 01 June 2003 , pp. e60-e60(1)

Publisher: Oxford University Press

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Abstract:

Construction methodologies for cDNA microarrays lack the ability to determine array integrity prior to hybridization, leaving the array itself a source of uncontrolled experimental variation. We solved this problem through development of a three-color cDNA array platform whereby printed probes are tagged with fluorescein and are compatible with Cy3 and Cy5 target labeling dyes when using confocal laser scanners possessing narrow bandwidths. Here we use this approach to: (i) develop a tracking system to monitor the printing of probe plates at predicted coordinates; (ii) define the quantity of immobilized probe necessary for quality hybridized array data to establish pre-hybridization array selection criteria; (iii) investigate factors that influence probe availability for hybridization; and (iv) explore the feasibility of hybridized data filtering using element fluorescein intensity. A direct and significant relationship (R² = 0.73, P < 0.001) between pre-hybridization average fluorescein intensity and subsequent hybridized replicate consistency was observed, illustrating that data quality can be improved by selecting arrays that meet defined pre-hybridization criteria. Furthermore, we demonstrate that our three-color approach provides a means to filter spots possessing insufficient bound probe from hybridized data sets to further improve data quality. Collectively, this strategy will improve microarray data and increase its utility as a sensitive screening tool.

Document Type: Research article

DOI: http://dx.doi.org/10.1093/nar/gng059

Affiliations: 1: The Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics, The Medical College of Wisconsin and Children’s Hospital of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA, 2: The Human and Molecular Genetics Center, 3: The Department of Pathology and

Publication date: 2003-06-01

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Nucleic Acids Research (NAR) is a fully Open Access journal, providing rapid publication of leading edge research into the nucleic acids under the following categories: chemistry, computational biology, genomics, molecular biology, nucleic acid enzymes, RNA and structural biology. There is a Survey and Summary section, and methods papers are published
in NAR Methods Online. Each year the first issue is devoted to biological databases, and a later issue to relevant web-based software resources.

Use of a three-color cDNA microarray platform to measure and control support-bound probe for improved data quality and reproducibility

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