Hi, I am working with Porcine Affymetrix in an experiment with technical replicates. I have 4 animals (2 control and 2 infected) and for each of them I have a technical replicate. Could somebody help me on how to include the technical replicates in a correct way in the model using eBayes analysis in the Limma package, please? I am not sure if the following script is correct: library(affy) Data.mes <- ReadAffy() phenoData(Data.mes) pd.mes <- data.frame(control = c(1,1,1,1,2,2,2,2), replica = c(1,1,2,2,3,3,4,4)) rownames(pd.mes) <- sampleNames(Data.mes) vl <- list(control = "1 is control, 2 is infected", replica = "1 is N1, 2 is N2, 3 is N3, and 4 is N4") phenoData(Data.mes) <- new("phenoData", pData = pd.mes, varLabels = vl) library(affyPLM) Data.mes.rma <- rma(Data.mes) e.mes.rma <- exprs(Data.mes.rma) library(limma) Data.mes.rma <- rma(Data.mes) design <- model.matrix(~factor(Data.mes.rma$control)+ Data.mes.rma$replica) fit <- lmFit(Data.mes.rma, design) ebayes <- eBayes(fit) tab <- topTable(ebayes, coef = 2, adjust = "fdr", n=24123) Thanks in advance, Lana Teixeira Fernandes PhD student Centre de Recerca en Sanitat Animal (CreSA) Barcelona - Spain Tel.: +34 93 581?45 61???? Fax: +34 93 581 44 90 lana.teixeira at cresa.uab.cat?????? www.cresa.es?