[BioC] Question on Limma technical replicate
Leleacquario at libero.it
Leleacquario at libero.it
Sun Jul 4 15:42:13 CEST 2010
Hi everyone,
I am trying to analyze an Agilent 4x44 experiment with the following
design:
> FileName Cy3 Cy5 time cell_line
1 M48R M48C 48 MCF7
2 M48C M48R 48 MCF7
3 M6R M6C 6 MCF7
4 M6C M6R 6 MCF7
5 D48R D48C 48 MDA
6 D48C D48R 48 MDA
7 D6R D6C 6 MDA
8 D6C D6R 6 MDA
where: M= MCF7 cell line while D=MDA cell line.
The number indicates the time and the final letter (C or D) indicates the
treatment (R) or control (C).
After normalization ecc ecc, I create my design file:
MCF748 <- grep("M48",targets$Cy3)
MCF76 <- grep("M6",targets$Cy3)
MDA48 <- grep("D48",targets$Cy3)
MDA6 <- grep("D6",targets$Cy3)
designMCF748 <- modelMatrix(targets[MCF748,],ref="M48C")
designMCF76 <- modelMatrix(targets[MCF76,],ref="M6C")
designMDA48 <- modelMatrix(targets[MDA48,],ref="D48C")
designMDA6 <- modelMatrix(targets[MDA6,],ref="D6C")
design <- blockDiag(designMCF748,designMCF76,designMDA48,designMDA6)
and then:
fit<- lmFit(MA, design, weights=NULL)
contrast.matrix <-makeContrasts(M48R,M6R,D48R,D6R, levels=design)
fit2<-contrasts.fit(fit,contrast.matrix)
efit<-eBayes(fit2)
Now..my problem is: how to consider the dye-swap arrays?
When I makeContrasts as above, it automatically excludes the swapped arrays.
Even trying with the function: "duplicateCorrelation", the output is "NA".
for all the values. I hope someone can help me!
Thanks in advance for any help.
Eleonora
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