Open Access Highly Accessed Methodology article

Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

Mathieu Clément-Ziza1, David Gentien2, Stanislas Lyonnet1, Jean-Paul Thiery23, Claude Besmond1 and Charles Decraene245*

Author Affiliations

1 INSERM U781, Université Paris Descartes, Faculté de Médecine, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, F-75015, Paris, France

2 Institut Curie, Département de Transfert, 26 rue d'ULM, F-75248, Paris cedex 05, France

3 IMCB Proteos, 61 Biopolis Drive, 138673, Singapore

4 Institut Curie, Centre de Recherche, 26 rue d'ULM, F-75248, Paris cedex 05, France

5 CNRS, UMR144, 26 rue d'ULM, F-75248, Paris cedex 05, France

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BMC Genomics 2009, 10:246  doi:10.1186/1471-2164-10-246

Published: 26 May 2009

Abstract

Background

For more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation.

Results

In this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity. The results demonstrate significant differences between these four methods.

Conclusion

In our hands, the WT-Ovation pico system proposed by Nugen appears to be the most suitable for RNA amplification. This comparative study will be useful to scientists needing to choose an amplification method to carry out microarray experiments involving samples comprising only a few cells and generating picogram amounts of RNA.