Dear List, I have a problem of estimability using LIMMA that I do not know how to solve. I think that I am using the right model, but I can not solve the problem so I need someone to help me out to figure out what is going on. I have an experiment with paired samples within two different strains. The data is as follows: Array mouse strain treatment 1 1 wt c 2 2 wt c 3 3 wt c 4 1 wt t 5 2 wt t 6 3 wt t 7 4 ko c 8 5 ko c 9 6 ko c 10 4 ko t 11 5 ko t 12 6 ko t It is exactly the same as the example 8.3 in the limma user guide, except that I have two strains, WT and KO with the same paired structure. I am using a linear model with the animal effect as a block effect to consider the pairing of the data, My code is the following: block=factor(c("A1","A2","A3","A1","A2","A3", "A4","A5","A6","A4","A5","A6"), levels=c("A1","A2","A3","A4","A5","A6") ) contrasts(block)=contr.sum(6) ttoEFF=factor(c("wtC","wtC","wtC","wtM","wtM","wtM", "koC","koC","koC","koM","koM","koM"), levels=c("wtC","wtM","koC","koM")) contrasts(ttoEFF)=contr.sum(4) design=model.matrix(~ -1 + ttoEFF + block) colnames(design) =c("wtC","wtM","koC","koM","A1","A2","A3","A4","A5") CM= makeContrasts(wtM-koM,wtC-koC,levels=design) fit=lmFit(eset,design) fit2=contrasts.fit(fit,CM) fit2=eBayes(fit2) DE=decideTests(fit2,method="nestedF",adjust.method="BY",p.value=0.05) >From " fit=lmFit(eset,design)" I get that the coefficient for the animal 5 is not estimable. I am considering the nested nature of the animals to its strain by the codification that I am using for the animals, and I have enough degrees of freedom to estimate the whole set of parameters of the model, so I do not understand why parameter A5 can not be estimated. If I work only in the arrays 1 to 6, considering only the strain WT separately from each other, the things work fine. For arrays 7 to 12 corresponding to the KO strain the things work ok as well , but if I extend the model to include the whole data, the parameter A5 corresponding to the animal 5 is not estimable and I don?t understand why. I want to use the whole data, because I am also interested in the comparisons between lines: CM= makeContrasts(wtC - wtM,koC-koM,levels=design) I will appreciate any help on this. Thank you very much. Pedro.- My session info is: > sessionInfo() Version 2.3.0 (2006-04-24) i386-pc-mingw32 attached base packages: [1] "splines" "tools" "methods" "stats" "graphics" "grDevices" "utils" "datasets" [9] "base" other attached packages: statmod qvalue MASS sma geneplotter affycoretools GOstats "1.2.4" "1.5.0" "7.2-27" "0.5.15" "1.10.0" "1.4.0" "1.6.0" Category hgu95av2 KEGG multtest genefilter survival xtable "1.4.0" "1.12.0" "1.12.0" "1.10.0" "1.10.0" "2.24" "1.3-1" RBGL GO graph Ruuid cluster codelink annotate "1.8.0" "1.12.0" "1.10.0" "1.10.0" "1.10.5" "1.0.0" "1.10.0" limma affy affyio Biobase "2.6.0" "1.10.0" "1.0.0" "1.10.0"