Nucleic Acids Research, 2000, Vol. 28, No. 22 4552-4557
© 2000 Oxford University Press
Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays
Department of Molecular Biology and Genomics and 1Department of Infectious Diseases, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
To examine the utility and performance of 50mer oligonucleotide (oligonucleotide probe) microarrays, gene-specific oligonucleotide probes were spotted along with PCR probes onto glass microarrays and the performance of each probe type was evaluated. The specificity of oligonucleotide probes was studied using target RNAs that shared various degrees of sequence similarity. Sensitivity was defined as the ability to detect a 3-fold change in mRNA. No significant difference in sensitivity between oligonucleotide probes and PCR probes was observed and both had a minimum reproducible detection limit of 
10 mRNA copies/cell. Specificity studies showed that for a given oligonucleotide probe any ‘non-target’ transcripts (cDNAs) >75% similar over the 50 base target may show cross-hybridization. Thus non-target sequences which have >75–80% sequence similarity with target sequences (within the oligonucleotide probe 50 base target region) will contribute to the overall signal intensity. In addition, if the 50 base target region is marginally similar, it must not include a stretch of complementary sequence >15 contiguous bases. Therefore, knowledge about the target sequence, as well as its similarity to other mRNAs in the target tissue or RNA sample, is required to design successful oligonucleotide probes for quality microarray results. Together these results validate the utility of oligonucleotide probe (50mer) glass microarrays.
* To whom correspondence should be addressed at present address: Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA. Tel: +1 734 622 1782; Fax: +1 734 622 5970; Email: steven.madore@pfizer.com Present address: Michael D. Kane, Genomic Solutions Inc., 4355 Varsity Drive East, Ann Arbor, MI 48108, USA
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