Research article

Assessing probe-specific dye and slide biases in two-color microarray data

Ruixiao Lu¹ , Geun-Cheol Lee² , Michael Shultz³ , Chris Dardick⁴ , Kihong Jung⁴ , Jirapa Phetsom⁴ , Yi Jia⁵ , Robert H Rice⁶ , Zelanna Goldberg⁷ , Patrick S Schnable⁵ , Pamela Ronald⁴ and David M Rocke⁸

¹Department of Data Analysis and Algorithm, Affymetrix, Inc., Santa Clara, California, USA

²College of Business Administration, Konkuk University, Korea

³Department of Molecular Biosciences, University of California, Davis, California, USA

⁴Department of Plant Pathology, University of California, Davis, California, USA

⁵Center for Plant Genomics, Iowa State University, Ames, Iowa, USA

⁶Department of Environmental Toxicology, University of California, Davis, California, USA

⁷Department of Radiation Oncology, University of California, Davis, Cancer Center, Sacramento, California, USA

⁸Division of Biostatistics, University of California, Davis, California, USA

author email corresponding author email

BMC Bioinformatics 2008, 9:314doi:10.1186/1471-2105-9-314

Published:	19 July 2008

Abstract

Background

A primary reason for using two-color microarrays is that the use of two samples labeled with different dyes on the same slide, that bind to probes on the same spot, is supposed to adjust for many factors that introduce noise and errors into the analysis. Most users assume that any differences between the dyes can be adjusted out by standard methods of normalization, so that measures such as log ratios on the same slide are reliable measures of comparative expression. However, even after the normalization, there are still probe specific dye and slide variation among the data. We define a method to quantify the amount of the dye-by-probe and slide-by-probe interaction. This serves as a diagnostic, both visual and numeric, of the existence of probe-specific dye bias. We show how this improved the performance of two-color array analysis for arrays for genomic analysis of biological samples ranging from rice to human tissue.

Results

We develop a procedure for quantifying the extent of probe-specific dye and slide bias in two-color microarrays. The primary output is a graphical diagnostic of the extent of the bias which called ECDF (Empirical Cumulative Distribution Function), though numerical results are also obtained.

Conclusion

We show that the dye and slide biases were high for human and rice genomic arrays in two gene expression facilities, even after the standard intensity-based normalization, and describe how this diagnostic allowed the problems causing the probe-specific bias to be addressed, and resulted in important improvements in performance. The R package LMGene which contains the method described in this paper has been available to download from Bioconductor.