Nucleic Acids Research Advance Access originally published online on June 12, 2007
Nucleic Acids Research 2007 35(12):4154-4163; doi:10.1093/nar/gkm348
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Nucleic Acids Research, 2007, Vol. 35, No. 12 4154-4163
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Explaining differences in saturation levels for Affymetrix GeneChip® arrays
1Department of Human Genetics, University of California Los Angeles, 2Department of Pathology, Keck School of Medicine, 3Molecular and Computational Biology Program, Department of Biological Sciences, 4Childrens Hospital Los Angeles, Department of Pathology and Laboratory Medicine, University of Southern California, Los Angeles, CA, USA and 5Department of Oncology, University of Cambridge, Cambridge, UK
*To whom correspondence should be addressed. Tel: 213 281 2010; Fax: 213 740 8631; Email: abdueva{at}usc.edu
Received August 7, 2006. Revised April 22, 2007. Accepted April 22, 2007.
The experimental spike-in studies of microarray hybridization conducted by Affymetrix demonstrate a nonlinear response of fluorescence intensity signal to target concentration. Several theoretical models have been put forward to explain these data. It was shown that the Langmuir adsorption isotherm recapitulates a general trend of signal response to concentration. However, this model fails to explain some key properties of the observed signal. In particular, according to the simple Langmuir isotherm, all probes should saturate at the same intensity level. However, this effect was not observed in the publicly available Affymetrix spike-in data sets. On the contrary, it was found that the saturation intensities vary greatly and can be predicted based on the probe sequence composition. In our experimental study, we attempt to account for the unexplained variation in the observed probe intensities using customized fluidics scripts. We explore experimentally the effect of the stringent wash, target concentration and hybridization time on the final microarray signal. The washing effect is assessed by scanning chips both prior to and after the stringent wash. Selective labeling of both specific and non-specific targets allows the visualization and investigation of the washing effect for both specific and non-specific signal components. We propose a new qualitative model of the probe-target hybridization mechanism that is in agreement with observed hybridization and washing properties of short oligonucleotide microarrays. This study demonstrates that desorption of incompletely bound targets during the washing cycle contributes to the observed difference in saturation levels.
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