Hello, My name is Vinicius Ribeiro and I?m a PhD student in University of S?o Paulo (Brazil). I would like to solve a doubt regarding my microarray experiment that I carried out. I used a comparison between wild type and mutant Listeria monocytogenes (bacteria) in stress conditions. Below you can find the sum of the commands that I used in my analysis. I had some warning messages and in the end of the processes I can not see p adj value <0.05 even with a lot of green and red spots in my slides (cy3 and cy5). I perfomed a similar experiment but using slides with 4 replicates of each probe and got excelent results but now using this slide (2 reps) I can not analyse properly. R commands > library(limma) > library(statmod) > targets.J1andC6<-readTargets("Targets_VR_2012.txt") > raw.J1andC6<-read.maimages(targets.J1andC6, source="genepix", wt.fun=wtflags(0)) Read Slide 75_913_0700.gpr Read Slide 76_913_0700.gpr Read Slide 83_835_0656.gpr > sorted.raw.J1andC6<-raw.J1andC6[order(raw.J1andC6$genes[,"Name"]),] > norm.J1andC6<-normalizeWithinArrays(sorted.raw.J1andC6, method="printtiploess",bc.method="none") > plotMA3by2(norm.J1andC6) > design=c(-1,-1,1) > corfit<-duplicateCorrelation(norm.J1andC6,design,ndup=2,spacing=1) > corfit$consensus [1] 0.7557859 > fit=lmFit(norm.J1andC6,design,ndup=2,correlation=corfit$consensus) > fit2<-eBayes(fit) Warning Message: Zero sample variances detected, have been offset > topTable(fit2,number=100,adjust="BH") Block Row Column ID Name logFC AveExpr t 1210 8 15 5 LM00001775 lmo2097 1.0534570 12.33612 4.180630 1161 4 15 3 LM00002616 lmo1999 1.2559634 11.25120 4.019180 378 4 4 13 LM00002219 lmo0433 1.9634563 12.87347 3.340647 Continue... P.Value adj.P.Val B 1210 0.0008789322 1 -4.286804 1161 0.0016090372 1 -4.328428 378 0.0047104593 1 -4.359437 Continue... > write.fit(fit2,file="J1andC6fit.txt",adjust="BH") I don?t know if this helps but attached you can find my raw data. I used GenePix to scan. Could anyone help me please. Is there other analysis that I can try? Thank you so much. Vinicius -------------- next part -------------- An embedded and charset-unspecified text was scrubbed... Name: Targets_VR_2012_Experimento Mar?o_3 reps.txt URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20120410="" 8e748f81="" attachment.txt="">