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Nucleic Acids Research 2005 33(6):e56; doi:10.1093/nar/gni054
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Published online 30 March 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

Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces

Sandrine Imbeaud*, Esther Graudens, Virginie Boulanger, Xavier Barlet, Patrick Zaborski, Eric Eveno, Odilo Mueller1, Andreas Schroeder1 and Charles Auffray

Array s/IMAGE, Genexpress, Functional Genomics and Systems Biology for Health LGN–UMR 7091, CNRS and Pierre and Marie Curie University of Paris 6, 7, rue Guy Môquet, 94801 Villejuif, France 1Agilent Technologies Waldbronn, Germany

*To whom correspondence should be addressed. Tel: +33 0 1 49 58 35 13; Fax: +33 0 1 49 58 35 09; Email: imbeaud{at}vjf.cnrs.fr

Received January 14, 2005. Revised March 2, 2005. Accepted March 2, 2005.

While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data.


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