Identification of an in vitro transcription-based artifact affecting oligonucleotide microarrays

This study identified the widely used T7 in vitro transcription system as a major source of artifact in the tiling array data from nine eukaryotic genomes. The most affected probes contained a sequence motif complementary to the +1 to +9 initial transcribed sequence (ITS) of the T7-(dT)24 primer. The abundance of 5′ ITS cRNA fragments produced during target preparation was sufficient to drive undesirable hybridization. A new T7-(dT)24 primer with a modified ITS was designed that shifts the artifactual motifs as predicted and reduces the effect of the artifact. A computational algorithm was generated to filter out the likely artifactual probes from existing whole-genome tiling array data and improve probe selection. Further studies of Arabidopsis thaliana were conducted using both T7-(dT)24 primers. While the artifact affected transcript discovery with tiling arrays, it showed only a minor impact on measurements of gene expression using commercially available ‘gene-only’ expression arrays.