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Nucleic Acids Research Advance Access published online on September 26, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm683
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D

Fabiana Perocchi, Zhenyu Xu, Sandra Clauder-Münster and Lars M. Steinmetz*

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

*To whom correspondence should be addressed. Tel: +49 6221 387-389; Fax: +49 6221 387-518; Email: larsms{at}embl.de

Received June 6, 2007. Revised August 20, 2007. Accepted August 20, 2007.

Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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