Hi all, I'm new to 2-colour arrays and I've been reading with interest a few papers about dye-bias: Dobbin, K., Shih, J.H. & Simon, R., 2003. Statistical design of reverse dye microarrays. Bioinformatics, 19(7), 803-810. Dombkowski, A.A. et al., 2004. Gene-specific dye bias in microarray reference designs. FEBS Letters, 560(1-3), 120-124. Kerr, M.K., 2003. Design Considerations for Efficient and Effective Microarray Studies. Biometrics, 59(4), 822-828. Martin-Magniette, M. et al., 2005. Evaluation of the gene-specific dye bias in cDNA microarray experiments. Bioinformatics, 21(9), 1995-2000. I've also been thinking about self-self hybridisations (the same sample hybridised to both Cy3 and Cy5): Fang, H. et al., 2007. Self-self Hybridization As An Alternative Experiment Design to Dye Swap for Two-color Microarrays. OMICS: A Journal of Integrative Biology, 11(1), 14. In the absence of any gene-dye interaction, the Cy3/Cy5 ratio should be zero in the absence of gene*dye interaction. I'm designing how best to hybridise our samples to Agilent chips. Our experiment is simply a comparison between two groups with 5 biological replicates per group (all 10 samples are biologically independent). From my reading, a multi-dye-swap and loop design both have the same precision to detect differences between the 2 groups using the same number of arrays. However, both are 4 time more precise (at best) than a reference design as relative abundances are measured directly, rather than indirectly. I'm opting for the multi-dye-swap where group 1 rep 1 is dye-swapped with group 2 rep 1, group 1 rep 2 is dye-swapped with group 2 rep 2 etc. This is because it is more robust in terms of missing data that may occur. Am I also right in thinking that it could be extended more easily than a loop design in the future - i.e. add additional biological replicates at a future date? The idea behind the dye-swap is to get a measure of the error variance due to dye-bias. I was wondering if were possible to obtain a better estimate by using self-self hybridisations and if so, how to code this into limma. For instance, my target file might look like: FileName Cy3 Cy5 1 G1 G2 2 G2 G1 3 G1 G2 4 G2 G1 5 G1 G2 6 G2 G1 7 G1 G2 8 G2 G1 9 G1 G2 10 G2 G1 Where G1 are biological replicates for Group 1 and G2 are biological replicates for Group 2. The same two RNA samples are dye-swapped e.g. array 1 and 2 are dye-swaps of the same RNA. Could I add, or even replace the dye-swaps, with self-self hybs to provide a better estimate the dye-bias like this: FileName Cy3 Cy5 1 G1 G2 2 G2 G1 3 G1 G2 4 G2 G1 5 G1 G2 6 G2 G1 7 G1 G2 8 G2 G1 9 G1 G2 10 G2 G1 11 G1 G1 12 G2 G2 I'd appreciate thoughts and comments if you have them! Cheers, Nathan -- -------------------------------------------------------- Dr. Nathan S. Watson-Haigh OCE Post Doctoral Fellow CSIRO Livestock Industries Queensland Bioscience Precinct St Lucia, QLD 4067 Australia Tel: +61 (0)7 3214 2922 Fax: +61 (0)7 3214 2900 Web: http://www.csiro.au/people/Nathan.Watson-Haigh.html