Open Access Open Badges Methodology article

Spotting effect in microarray experiments

Tristan Mary-Huard1*, Jean-Jacques Daudin1, Stéphane Robin1, Frédérique Bitton2, Eric Cabannes3 and Pierre Hilson24

Author Affiliations

1 Institut National Agronomique Paris-Grignon, 16 rue Claude Bernard, 75231 Paris, France

2 UMR Génomique Végétale, INRA-CNRS-Université d'Evry, CP 5708, F-91057 Evry, France

3 Laboratoire d'Immunologie Virale, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France

4 Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent, Belgium

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BMC Bioinformatics 2004, 5:63  doi:10.1186/1471-2105-5-63

Published: 19 May 2004



Microarray data must be normalized because they suffer from multiple biases. We have identified a source of spatial experimental variability that significantly affects data obtained with Cy3/Cy5 spotted glass arrays. It yields a periodic pattern altering both signal (Cy3/Cy5 ratio) and intensity across the array.


Using the variogram, a geostatistical tool, we characterized the observed variability, called here the spotting effect because it most probably arises during steps in the array printing procedure.


The spotting effect is not appropriately corrected by current normalization methods, even by those addressing spatial variability. Importantly, the spotting effect may alter differential and clustering analysis.