Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes. Nucleic Acids Res 2003 Feb 15;31(4):e14
Date
02/13/2003Pubmed ID
12582259Pubmed Central ID
PMC150246DOI
10.1093/nar/gng014Scopus ID
2-s2.0-18144448930 (requires institutional sign-in at Scopus site) 53 CitationsAbstract
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.
Author List
Hessner MJ, Wang X, Hulse K, Meyer L, Wu Y, Nye S, Guo SW, Ghosh SAuthor
Martin J. Hessner PhD Professor in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
CarbocyaninesDNA Probes
DNA, Complementary
Fluorescein
Humans
Oligonucleotide Array Sequence Analysis
Reproducibility of Results
Sensitivity and Specificity
