title: Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes.
abstract: Gene expression studies using microarrays have great potential to generate new insights into human disease pathogenesis, but data quality remains a major obstacle. In particular, there does not exist a method to determine prior to hybridization whether an array will yield high quality data, given good study design and target preparation. We have solved this problem through development of a three-color cDNA microarray platform where printed probes are fluorescein labeled, but are spectrally compatible with Cy3 and Cy5 dye-labeled targets when using confocal laser scanners possessing narrow bandwidths. This approach enables prehybridization evaluation of array/spot morphology, DNA deposition and retention and background levels. By using these measurements and the intra-slide coefficient of variation for fluorescence intensity we show that slides in the same batch are not equivalent and measurable prehybridization parameters can be predictive of hybridization performance as determined by replicate consistency. When hybridizing target derived from two cell lines to high and low quality replicate pairs (n = 50 pairs), a direct and significant relationship between prehybridization signal-to-background noise and post-hybridization reproducibility (R2 = 0.80, P < 0.001) was observed. We therefore conclude that slide selection based upon prehybridization quality scores will greatly benefit the ability to generate reliable gene expression data.
authors.affiliation: The Max McGee National Research Center for Juvenile Diabetes, The Medical College of Wisconsin and Children's Hospital of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. mhessner@mcw.edu
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Hessner
authors.forename: Xujing
authors.initials: X
authors.lastname: Wang
authors.forename: Katie
authors.initials: K
authors.lastname: Hulse
authors.forename: Lisa
authors.initials: L
authors.lastname: Meyer
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.forename: Steven
authors.initials: S
authors.lastname: Nye
authors.forename: Sun-Wei
authors.initials: SW
authors.lastname: Guo
authors.forename: Soumitra
authors.initials: S
authors.lastname: Ghosh
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D002232:Carbocyanines; D015342:DNA Probes; D018076:DNA, Complementary; C089187:cyanine dye 3; C085321:cyanine dye 5; D019793:Fluorescein