Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays.
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.
Wang
Hong-Ying
HY
The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA.
Malek
Renae L
RL
Kwitek
Anne E
AE
Greene
Andrew S
AS
Luu
Truong V
TV
Behbahani
Babak
B
Frank
Bryan
B
Quackenbush
John
J
Lee
Norman H
NH
D017360:Arabidopsis / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D015345:Oligonucleotide Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D015345:Oligonucleotide Probes