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

Content Provider	Springer Nature : BioMed Central
Author	Hessner, Martin J Singh, Vineet K Wang, Xujing Khan, Shehnaz Tschannen, Michael R Zahrt, Thomas C
Abstract	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.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-5-12.pdf
Ending Page	11
Page Count	11
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/1471-2164-5-12
Journal	BMC Genomics
Issue Number	1
Volume Number	5
Language	English
Publisher	BioMed Central
Publisher Date	2004-02-09
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics Spotted oligonucleotide arrays 70-mers gene expression analysis
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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