Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays
<p>Abstract</p> <p>Background</p> <p>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 previo...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2004-02-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://www.biomedcentral.com/1471-2164/5/12 |
| _version_ | 1828490329605537792 |
|---|---|
| author | Khan Shehnaz Wang Xujing Singh Vineet K Hessner Martin J Tschannen Michael R Zahrt Thomas C |
| author_facet | Khan Shehnaz Wang Xujing Singh Vineet K Hessner Martin J Tschannen Michael R Zahrt Thomas C |
| author_sort | Khan Shehnaz |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 <it>Staphylococcus aureus</it>-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a <it>Mycobacterium tuberculosis </it>oligonucleotide microarray.</p> <p>Results</p> <p>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 <it>M. tuberculosis </it>H37Rv and <it>M. tuberculosis </it>mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R<sup>2 </sup>= 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 μM to 0.78 μM) in the presence of 0.5 μM 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).</p> <p>Conclusions</p> <p>This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.</p> |
| first_indexed | 2024-12-11T10:42:46Z |
| format | Article |
| id | doaj.art-b66a839703b64389b7c197eab6ef667c |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-11T10:42:46Z |
| publishDate | 2004-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-b66a839703b64389b7c197eab6ef667c2022-12-22T01:10:33ZengBMCBMC Genomics1471-21642004-02-01511210.1186/1471-2164-5-12Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arraysKhan ShehnazWang XujingSingh Vineet KHessner Martin JTschannen Michael RZahrt Thomas C<p>Abstract</p> <p>Background</p> <p>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 <it>Staphylococcus aureus</it>-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a <it>Mycobacterium tuberculosis </it>oligonucleotide microarray.</p> <p>Results</p> <p>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 <it>M. tuberculosis </it>H37Rv and <it>M. tuberculosis </it>mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R<sup>2 </sup>= 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 μM to 0.78 μM) in the presence of 0.5 μM 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).</p> <p>Conclusions</p> <p>This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.</p>http://www.biomedcentral.com/1471-2164/5/12Spotted oligonucleotide arrays70-mersgene expression analysis |
| spellingShingle | Khan Shehnaz Wang Xujing Singh Vineet K Hessner Martin J Tschannen Michael R Zahrt Thomas C Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays BMC Genomics Spotted oligonucleotide arrays 70-mers gene expression analysis |
| title | Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays |
| title_full | Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays |
| title_fullStr | Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays |
| title_full_unstemmed | Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays |
| title_short | Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays |
| title_sort | utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70 mer arrays |
| topic | Spotted oligonucleotide arrays 70-mers gene expression analysis |
| url | http://www.biomedcentral.com/1471-2164/5/12 |
| work_keys_str_mv | AT khanshehnaz utilizationofalabeledtrackingoligonucleotideforvisualizationandqualitycontrolofspotted70merarrays AT wangxujing utilizationofalabeledtrackingoligonucleotideforvisualizationandqualitycontrolofspotted70merarrays AT singhvineetk utilizationofalabeledtrackingoligonucleotideforvisualizationandqualitycontrolofspotted70merarrays AT hessnermartinj utilizationofalabeledtrackingoligonucleotideforvisualizationandqualitycontrolofspotted70merarrays AT tschannenmichaelr utilizationofalabeledtrackingoligonucleotideforvisualizationandqualitycontrolofspotted70merarrays AT zahrtthomasc utilizationofalabeledtrackingoligonucleotideforvisualizationandqualitycontrolofspotted70merarrays |