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Open Access Methodology article

Comparing transformation methods for DNA microarray data

Helene H Thygesen* and Aeilko H Zwinderman

Author Affiliations

Clinical Epidemiology and Biostatistics, Academisch Medisch Centrum, University of Amsterdam, Meibergdreef 9, 1100 DD Amsterdam, Netherlands

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BMC Bioinformatics 2004, 5:77  doi:10.1186/1471-2105-5-77

Published: 17 June 2004



When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include subtraction of an estimated background signal, subtracting the reference signal, smoothing (to account for nonlinear measurement effects), and more. Different authors use different approaches, and it is generally not clear to users which method they should prefer.


We used the ratio between biological variance and measurement variance (which is an F-like statistic) as a quality measure for transformation methods, and we demonstrate a method for maximizing that variance ratio on real data. We explore a number of transformations issues, including Box-Cox transformation, baseline shift, partial subtraction of the log-reference signal and smoothing. It appears that the optimal choice of parameters for the transformation methods depends on the data. Further, the behavior of the variance ratio, under the null hypothesis of zero biological variance, appears to depend on the choice of parameters.


The use of replicates in microarray experiments is important. Adjustment for the null-hypothesis behavior of the variance ratio is critical to the selection of transformation method.