I think there is a bug on read.maimages limma function. I try: > targets <- readTargets() > files <- targets[,c("FileNameCy3","FileNameCy5")] > RG <- read.maimages(files, source="imagene") Read header information Read lamina1Cy3_551.txt Erro em `[.data.frame`(obj, , columns$f) : undefined columns selected After a lot of time, I try: > targets <- readTargets() > files <- targets[,c("FileNameCy3","FileNameCy5")] > RG <- read.maimages(files, source="imagene",columns=list(f="Signal Median",b="Background Median")) Read header information Read lamina1Cy3_551.txt Read lamina1Cy5_650.txt Read lamina2Cy3_551.txt Read lamina2Cy5_650.txt Read lamina3Cy3_532.txt Read lamina3Cy5_650.txt Read lamina5Cy3_532.txt Read lamina5Cy5_635.txt > Here are more information > targets SlideNumber FileNameCy3 FileNameCy5 Cy3 Cy5 1 1 lamina1Cy3_551.txt lamina1Cy5_650.txt Pra Banha 2 2 lamina2Cy3_551.txt lamina2Cy5_650.txt Pra Banha 3 3 lamina3Cy3_532.txt lamina3Cy5_650.txt Pra Banha 4 5 lamina5Cy3_532.txt lamina5Cy5_635.txt Pra Banha > files FileNameCy3 FileNameCy5 1 lamina1Cy3_551.txt lamina1Cy5_650.txt 2 lamina2Cy3_551.txt lamina2Cy5_650.txt 3 lamina3Cy3_532.txt lamina3Cy5_650.txt 4 lamina5Cy3_532.txt lamina5Cy5_635.txt > Header of my files Begin Header version 8.0.1 Date Thu Jun 11 12:35:08 BRT 2009 Image File C:\Users\[snip]\lamina1Cy3_551.tif Page 0 Page Name Inverted false Begin Field Dimensions Field Metarows Metacols Rows Cols A 2 2 32 24 B 2 2 32 24 End Field Dimensions Begin Measurement parameters Segmentation Method auto Signal Low 0.0 Signal High 0.0 Background Low 0.0 Background High 0.0 Background Buffer 2.0 Background Width 5.0 End Measurement parameters Begin Alerts Control Type Minimum threshold If tested Percentage allowed If failed Maximum threshold If tested Percentage allowed If failed CV threshold If tested If failed BLANK 0.0 false 1.0% false 500.0 true 0.1% false 1.0 false false POSITIVE 1000.0 true 0.1% false 100000.0 false 1.0% false 1.0 false false End Alerts Begin Quality Flags Begin Flagging Settings Empty Spots true Threshold: 0.96 Poor Spots true Begin Poor Spots Parameters Background contamination flag true Threshold: 0.9995 Background tested against subgrid data only true Signal contamination flag true Threshold: 0.9995 Signal contamination test connected to background contamination threshold true Ignored percentage flag true Threshold: 25.2 Open perimeter flag true Threshold: 20.0 Shape regularity flag true Threshold: 0.6 Area To Perimeter Ratio flag false Threshold: 0.7 Offset flag true Threshold: 60.0 Saturation flag true End Poor Spots Parameters Negative Spots true End Flagging Settings Begin Flagged spots # of Empty Spots: 2667 # of Poor Spots: 288 # of Negative Spots: 0 # of Manually Flagged Spots: 0 End Flagged spots End Quality Flags End Header Begin Raw Data Field Meta Row Meta Column Row Column Gene ID Annotation 1 Annotation 2 Flag Signal Median Background Median Signal Mode Background Mode Signal Area Background Area Signal Total Background Total Signal Stdev Background Stdev Shape Regularity Ignored Area Spot Area Ignored Median Area To Perimeter Open Perimeter XCoord YCoord Diameter Position offset Offset X Offset Y Expected X Expected Y CM-X CM-Y CM Offset CM Offset-X CM Offset-Y Min Diam Max Diam Control Failed Control Background contamination present Signal contamination present Ignored % failed Open perimeter failed Shape regularity failed Perim-to-area failed Offset failed Empty spot Negative spot Selected spot Saturated spot > sessionInfo() R version 2.9.2 (2009-08-24) i486-pc-linux-gnu locale: LC_CTYPE=pt_BR.UTF-8;LC_NUMERIC=C;LC_TIME=pt_BR.UTF-8;LC_COLLATE=pt_BR .UTF-8;LC_MONETARY=C;LC_MESSAGES=pt_BR.UTF-8;LC_PAPER=pt_BR.UTF-8;LC_N AME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=pt_BR.UTF-8;LC_IDENTI FICATION=C attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] limma_2.18.3 loaded via a namespace (and not attached): [1] tools_2.9.2 > Am I doing any mistake? Thank you very much -- Marcelo Luiz de Laia Universidade do Estado de Santa Catarina UDESC - www.cav.udesc.br Lages - SC - Brazil Linux user number 487797