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Open Access Highly Accessed Research article

A robust method for estimating gene expression states using Affymetrix microarray probe level data

Megu Ohtaki1*, Keiko Otani1, Keiko Hiyama2, Naomi Kamei3, Kenichi Satoh1 and Eiso Hiyama3

Author Affiliations

1 Department of Environmetrics and Biometrics, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan

2 Department of Translational Cancer Research, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan

3 Natural Science Center for Basic Research and Development, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan

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BMC Bioinformatics 2010, 11:183  doi:10.1186/1471-2105-11-183

Published: 12 April 2010



Microarray technology is a high-throughput method for measuring the expression levels of thousand of genes simultaneously. The observed intensities combine a non-specific binding, which is a major disadvantage with microarray data. The Affymetrix GeneChip assigned a mismatch (MM) probe with the intention of measuring non-specific binding, but various opinions exist regarding usefulness of MM measures. It should be noted that not all observed intensities are associated with expressed genes and many of those are associated with unexpressed genes, of which measured values express mere noise due to non-specific binding, cross-hybridization, or stray signals. The implicit assumption that all genes are expressed leads to poor performance of microarray data analyses. We assume two functional states of a gene - expressed or unexpressed - and propose a robust method to estimate gene expression states using an order relationship between PM and MM measures.


An indicator 'probability of a gene being expressed' was obtained using the number of probe pairs within a probe set where the PM measure exceeds the MM measure. We examined the validity of the proposed indicator using Human Genome U95 data sets provided by Affymetrix. The usefulness of 'probability of a gene being expressed' is illustrated through an exploration of candidate genes involved in neuroblastoma prognosis. We identified the candidate genes for which expression states differed (un-expressed or expressed) when compared between two outcomes. The validity of this result was subsequently confirmed by quantitative RT-PCR.


The proposed qualitative evaluation, 'probability of a gene being expressed', is a useful indicator for improving microarray data analysis. It is useful to reduce the number of false discoveries. Expression states - expressed or unexpressed - correspond to the most fundamental gene function 'On' and 'Off', which can lead to biologically meaningful results.