Evidence of cross-hybridization artifact in expressed sequence tags (ESTs) on cDNA microarrays

We present evidence of cross-hybridization artifact intrinsic to spotted single-dye cDNA microarrays as a result of cDNA containing 5’-end sequences of consecutive thymidine (dT) residues. These poly(dT) tracts result from the synthesis, via oligo (dT) primed reverse transcription, of expressed sequence tags (EST) cDNA from a polyadenylated mRNA template. Analysis of gene expression data from two separate experiments involving commercially available cDNA microarrays showed that ESTs whose reported sequences contain more than 11 consecutive 5’-end dT residues appeared to be strongly co-expressed. Our results suggest that expression data from microarray sequences containing 5’ poly(dT) tracts is likely to be due to cross-hybridization rather than the actual mRNA expression signal. This indicates that existing data generated by cDNA microarrays may be unreliable and should be £ltered to remove EST sequences containing signi£cant poly(dT) residues. Ideally, manufacturers of cDNA microarrays should excise any 5’ poly(dT) tract from cDNA prior to deposition on the substrate.