Faculty Collaboration Database

Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays. Genome Biol 2003;4(1):R5

Date

01/24/2003

Pubmed ID

12540297

Pubmed Central ID

PMC151289

DOI

10.1186/gb-2003-4-1-r5

Scopus ID

2-s2.0-18544410036 (requires institutional sign-in at Scopus site)   122 Citations

Abstract

BACKGROUND: Long oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear.

RESULTS: Unmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 microm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR.

CONCLUSIONS: Microarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.

Author List

Wang HY, Malek RL, Kwitek AE, Greene AS, Luu TV, Behbahani B, Frank B, Quackenbush J, Lee NH

Author

Anne E. Kwitek PhD Professor in the Physiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Arabidopsis
DNA, Complementary
Oligonucleotide Array Sequence Analysis
Oligonucleotide Probes
Polymerase Chain Reaction
Sensitivity and Specificity