Hello Everyone, I am trying to compare two red-labeled samples via a green-labeled reference RNA. Here is an example of a script I've run: > library(limma) > files <- dir(pattern="*.gpr") > RG <- read.maimages(files, source="genepix") Read 099.gpr Read 101.gpr Read 105.gpr Read 107.gpr Read 111.gpr Read 113.gpr > > targets <- readTargets() > targets FileName Cy3 Cy5 Name 1 099.gpr Ref A3 99 2 101.gpr Ref A5 101 3 105.gpr Ref A3 105 4 107.gpr Ref A5 107 5 111.gpr Ref A3 111 6 113.gpr Ref A5 113 > > RG$genes <- readGAL() > design <- designMatrix(targets,ref="Ref") > design A3 A5 1 1 0 2 0 1 3 1 0 4 0 1 5 1 0 6 0 1 My intention is to compare "A3" with "A5" via a common reference, "Ref", similar (I think) to the example in the limma User's guide section 8.2 "ApoAI Knockout Data ...". What am I doing wrong? Is my Targets.txt file set up incorrectly? In its current state it results in topTable giving me a list that seems appropriate for contrast between Cy3 and Cy5. I've also tried creating a "design" object like the one in the example manually by writing it in a text editor and importing it with read.table(). I just get nonsense back from that. I'm running BioC v1.3 R 1.8.1 on linux kernel 2.6.4 Thanks in advance for the help. -Dennis

You need to define a contrast matrix. See section 7.3 of the limma User's Guide. Gordon >Hello Everyone, >I am trying to compare two red-labeled samples via a green-labeled >reference RNA. Here is an example of a script I've run: > > > library(limma) > > files <- dir(pattern="*.gpr") > > RG <- read.maimages(files, source="genepix") >Read 099.gpr >Read 101.gpr >Read 105.gpr >Read 107.gpr >Read 111.gpr >Read 113.gpr > > > > targets <- readTargets() > > targets > FileName Cy3 Cy5 Name >1 099.gpr Ref A3 99 >2 101.gpr Ref A5 101 >3 105.gpr Ref A3 105 >4 107.gpr Ref A5 107 >5 111.gpr Ref A3 111 >6 113.gpr Ref A5 113 > > > > RG$genes <- readGAL() > > design <- designMatrix(targets,ref="Ref") > > design > A3 A5 >1 1 0 >2 0 1 >3 1 0 >4 0 1 >5 1 0 >6 0 1 > >My intention is to compare "A3" with "A5" via a common reference, "Ref", >similar (I think) to the example in the limma User's guide section 8.2 >"ApoAI Knockout Data ...". What am I doing wrong? Is my Targets.txt file >set up incorrectly? In its current state it results in topTable giving >me a list that seems appropriate for contrast between Cy3 and Cy5. I've >also tried creating a "design" object like the one in the example >manually by writing it in a text editor and importing it with >read.table(). I just get nonsense back from that. > >I'm running BioC v1.3 R 1.8.1 on linux kernel 2.6.4 > >Thanks in advance for the help. > >-Dennis >