Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays

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• 作者：Martin J Hessner (1) (2)
  Vineet K Singh (3)
  Xujing Wang (1) (2)
  Shehnaz Khan (2)
  Michael R Tschannen (2)
  Thomas C Zahrt (3)
  
• 关键词：Spotted oligonucleotide arrays ; 70 ; mers ; gene expression analysis
• 刊名：BMC Genomics
• 出版年：2004
• 出版时间：December 2004
• 年：2004
• 卷：5
• 期：1
• 全文大小：3616KB
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• 作者单位：Martin J Hessner (1) (2)
  Vineet K Singh (3)
  Xujing Wang (1) (2)
  Shehnaz Khan (2)
  Michael R Tschannen (2)
  Thomas C Zahrt (3)
  
  1. 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, 53226, Milwaukee, WI, USA
  2. The Human and Molecular Genetics Center, The Medical College of Wisconsin, 8701 Watertown Plank Road, 53226, Milwaukee, WI, USA
  3. Department of Microbiology and Molecular Genetics, The Medical College of Wisconsin, 8701 Watertown Plank Road, 53226, Milwaukee, WI, USA
  

文摘

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 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). 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.