A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina
1 Univ Paris-Sud 11, Institut de Génétique et Microbiologie UMR8621, F- 91405 Orsay, France
2 CNRS, Institut de Génétique et Microbiologie, UMR8621, F-91404 Orsay, France
3 Univ Paris-Sud 11, F- 91405 Orsay, France
4 CNRS, Centre de Génétique Moléculaire, FRE3144, GODMAP, F-91190 Gif sur Yvette, France
5 UFR des Sciences du Vivant, Université de Paris 7 - Denis Diderot, F-75205 Paris CEDEX 13, France
6 Université Victor Segalen, Bordeaux 2, Institut de Biochimie et Génétique Cellulaires, UMR 5095, 1 rue Camille Saint Saëns, F-33077 Bordeaux cedex, France
7 INSERM, Génomique Fonctionnelle des tumeurs solides, UMR U-674, IUH, Université Paris-Descartes, Paris, F-75010, France
8 USR 3278 CNRS-EPHE CRIOBE-Université de Perpignan BP 1013 Papetoai Moorea 98729 Polynésie Française
BMC Research Notes 2010, 3:171 doi:10.1186/1756-0500-3-171Published: 18 June 2010
The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome.
We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS.
A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.