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Identification of an in vitro transcription‐based artifact affecting oligonucleotide microarrays
Author(s) -
Nelson David C.,
Wohlbach Dana J.,
Rodesch Matthew J.,
Stolc Viktor,
Sussman Michael R.,
Samanta Manoj P.
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.06.033
Subject(s) - computational biology , tiling array , dna microarray , biology , genome , oligonucleotide , primer (cosmetics) , genetics , gene , gene expression , chemistry , organic chemistry
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.