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Nucleic Acids Research 2005 33(3):e29; doi:10.1093/nar/gni029
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Published online 17 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays

Joerg Schlingemann, Olaf Thuerigen, Carina Ittrich1, Grischa Toedt, Heidi Kramer, Meinhard Hahn* and Peter Lichter

Division of Molecular Genetics, Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 1 Central Unit Biostatistics, Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

*To whom correspondence should be addressed. Tel: +49 6221 424677; Fax: +49 6221 424639; Email: m.hahn{at}dkfz.de

Received November 11, 2004. Revised January 23, 2005. Accepted January 23, 2005.

Gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, we introduce a novel protocol in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA. We demonstrate high fidelity in arrays using up to 105-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. These results demonstrate that our protocol describes an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is the limited source material.


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