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Open Access Highly Accessed Methodology article

Separate-channel analysis of two-channel microarrays: recovering inter-spot information

Gordon K Smyth12 and Naomi S Altman3*

Author Affiliations

1 Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic 3052, Australia

2 Department of Mathematics and Statistics, University of Melbourne, Vic 3010, Australia

3 Department of Statistics, The Pennsylvania State University, University Park, PA 16802–2111, USA

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BMC Bioinformatics 2013, 14:165  doi:10.1186/1471-2105-14-165

Published: 26 May 2013



Two-channel (or two-color) microarrays are cost-effective platforms for comparative analysis of gene expression. They are traditionally analysed in terms of the log-ratios (M-values) of the two channel intensities at each spot, but this analysis does not use all the information available in the separate channel observations. Mixed models have been proposed to analyse intensities from the two channels as separate observations, but such models can be complex to use and the gain in efficiency over the log-ratio analysis is difficult to quantify. Mixed models yield test statistics for the null distributions can be specified only approximately, and some approaches do not borrow strength between genes.


This article reformulates the mixed model to clarify the relationship with the traditional log-ratio analysis, to facilitate information borrowing between genes, and to obtain an exact distributional theory for the resulting test statistics. The mixed model is transformed to operate on the M-values and A-values (average log-expression for each spot) instead of on the log-expression values. The log-ratio analysis is shown to ignore information contained in the A-values. The relative efficiency of the log-ratio analysis is shown to depend on the size of the intraspot correlation. A new separate channel analysis method is proposed that assumes a constant intra-spot correlation coefficient across all genes. This approach permits the mixed model to be transformed into an ordinary linear model, allowing the data analysis to use a well-understood empirical Bayes analysis pipeline for linear modeling of microarray data. This yields statistically powerful test statistics that have an exact distributional theory. The log-ratio, mixed model and common correlation methods are compared using three case studies. The results show that separate channel analyses that borrow strength between genes are more powerful than log-ratio analyses. The common correlation analysis is the most powerful of all.


The common correlation method proposed in this article for separate-channel analysis of two-channel microarray data is no more difficult to apply in practice than the traditional log-ratio analysis. It provides an intuitive and powerful means to conduct analyses and make comparisons that might otherwise not be possible.

Loop design; Unconnected design; Reference design; Intraclass correlation; False discovery rate; Power; Efficiency