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


CONCLUSIONS
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.
Clément-Ziza
Mathieu
M
Institut Curie, Département de Transfert, 26 Rue d'ULM, F-75248, Paris cedex 05, France. mathieu.clement-ziza@biotec.tu-dresden.de
Gentien
David
D
Lyonnet
Stanislas
S
Thiery
Jean-Paul
JP
Besmond
Claude
C
Decraene
Charles
C
D002460:Cell Line; D020869:Gene Expression Profiling; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D012313:RNA