Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays. BMC Genomics 2004 Feb 09;5(1):12
Date
03/17/2004Pubmed ID
15018646Pubmed Central ID
PMC362869DOI
10.1186/1471-2164-5-12Scopus ID
2-s2.0-2442669076 (requires institutional sign-in at Scopus site) 33 CitationsAbstract
BACKGROUND: Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray.
RESULTS: Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 +/- 0.07), replicates lacking (0.74 +/- 0.10), or replicates with and without (0.70 +/- 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 microM to 0.78 microM) in the presence of 0.5 microM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression).
CONCLUSIONS: This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.
Author List
Hessner MJ, Singh VK, Wang X, Khan S, Tschannen MR, Zahrt TCAuthor
Martin J. Hessner PhD Professor in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Analysis of VarianceCarbocyanines
DNA, Bacterial
Gene Expression Profiling
Mycobacterium tuberculosis
Nucleic Acid Hybridization
Oligonucleotide Array Sequence Analysis
Oligonucleotide Probes
Quality Control
RNA, Bacterial
Reference Standards
Reproducibility of Results
Staphylococcus aureus
