[BioC] Question on Limma technical replicate

Leleacquario at libero.it Leleacquario at libero.it
Sun Jul 4 15:29:57 CEST 2010


Hi everyone, 
I am trying to analyze an   Agilent 4x44  experiment with the following 
design: 

 FileName  Cy3  Cy5 time cell_line
1 US45102933_251485026973_S01_GE2_105_Dec08_2_noSI_1_1.txt M48R M48C   48      
MCF7
2 US45102933_251485026973_S01_GE2_105_Dec08_2_noSI_1_2.txt M48C M48R   48      
MCF7
3 US45102933_251485026973_S01_GE2_105_Dec08_2_noSI_1_3.txt  M6R  M6C    6      
MCF7
4 US45102933_251485026975_S01_GE2_105_Dec08_2_noSI_1_2.txt  M6C  M6R    6      
MCF7
5 US45102933_251485026974_S01_GE2_105_Dec08_2_noSI_1_1.txt D48R D48C   
48       MDA
6 US45102933_251485026975_S01_GE2_105_Dec08_2_noSI_1_1.txt D48C D48R   
48       MDA
7 US45102933_251485026974_S01_GE2_105_Dec08_2_noSI_1_3.txt  D6R  D6C    
6       MDA
8 US45102933_251485026974_S01_GE2_105_Dec08_2_noSI_1_4.txt  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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