Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays.
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.
Hessner
Martin J
MJ
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. mhessner@mcw.edu
Singh
Vineet K
VK
Wang
Xujing
X
Khan
Shehnaz
S
Tschannen
Michael R
MR
Zahrt
Thomas C
TC
D000704:Analysis of Variance; D002232:Carbocyanines / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics* / Q000592:standards; D011786:Quality Control; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D013211:Staphylococcus aureus / Q000235:genetics
D002232:Carbocyanines; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; C089187:cyanine dye 3; C085321:cyanine dye 5