Dear BioConductor list: I was trying to compute spot weights with a couple of small test files by manually data reading. The computation was done without problem. But the results showed zero weight for each spot, even though most of them were expected to get weight one . Please see the function and result below. Thanks a bunch for any help! myfunRatio <- function(x, threshold=1.2) { okred <- abs(x[,"CH1I_MEAN"]/x[,"CH1B_MEDIAN"]) < threshold okgreen <- abs(x[,"CH2I_MEAN"]/x[,"CH2B_MEDIAN"]) < threshold as.numeric(okred & okgreen) } RG_ratioWeight <- read.maimages(file,columns=list(Rf="CH1I_MEAN",Gf="CH2I_MEAN",Rb="CH1B _MEDIAN",Gb="CH2B_MEDIAN"), wt.fun=myfunRatio) > RG_ratioWeight An object of class "RGList" $R testWeight testWeight1 [1,] 10 12 [2,] 11 13 [3,] 12 14 [4,] 13 15 [5,] 14 16 [6,] 15 17 [7,] 16 18 [8,] 17 19 $G testWeight testWeight1 [1,] 5.0 7.0 [2,] 6.0 8.0 [3,] 7.0 9.0 [4,] 8.0 10.0 [5,] 4.3 6.3 [6,] 2.0 4.0 [7,] 3.0 5.0 [8,] 4.0 6.0 $Rb testWeight testWeight1 [1,] 1.0 3.0 [2,] 10.0 12.0 [3,] 3.0 5.0 [4,] 2.0 4.0 [5,] 3.0 5.0 [6,] 2.5 4.5 [7,] 5.0 7.0 [8,] 2.0 4.0 $Gb testWeight testWeight1 [1,] 0.5 2.5 [2,] 1.5 3.5 [3,] 2.5 4.5 [4,] 3.5 5.5 [5,] 4.5 6.5 [6,] 1.0 3.0 [7,] 2.0 4.0 [8,] 5.0 7.0 $weights testWeight testWeight1 [1,] 0 0 [2,] 0 0 [3,] 0 0 [4,] 0 0 [5,] 0 0 [6,] 0 0 [7,] 0 0 [8,] 0 0 $targets FileName testWeight testWeight.txt testWeight1 testWeight1.txt Also I wanted to filter out some "bad" spot before I apply statistic tools, for example, paired t-test. Any suggestions about filtering? JP- xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx x Jianping Jin Ph.D. x x Bioinformatics scientist x x Center for bioinformatics x x 3133 Bioinformatics Building x x CB# 7104 x x University of North Carolina x x Chapel Hill, NC 27599 x x Tel: (919)843-6105 x x Fax: (919)843-3103 x x E-mail: jjin at email.unc.edu x xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx