
Dear all, 
I am trying analyze my 2-color microarray data with limma, and I am not sure which design I should use. I have total of 4 arrays:array 1 to 4. array 1 and array 2 are from the same biological experiment with dye swap. array 3 and 4 are from two individualexperiments and also dye swap. I am trying compare gene expression profiles between my mutant 1 (isp-1) and mutant2 (isp-1;ceh-23) For example:                    Cy3                                           Cy5array1           isp-1 (exp1)                            isp-1;ceh-23(exp1)array2           isp-1;ceh-23 (exp1)                 isp-1 (exp1)array3           isp-1 (exp2)                            isp-1;ceh-23(exp2)array4           isp-1;ceh-23 (exp3)                 isp-1 (exp3)I think what I have done is to find the coefficient for each biological experiment and take the average of the coefficients at the end, but I am not sure if this is the best way of doing it.Here's my script :
> library(limma)

> targets <- readTargets("targets.txt")

> RG <-
read.maimages(targets,source="agilent.median")

Read US83000170_252018610364_S01_GE2_105_Dec08_1_3.txt 

Read US83000170_252018610364_S01_GE2_105_Dec08_1_4.txt 

Read US83000170_252018610386_S01_GE2_105_Dec08_1_2.txt 

Read US83000170_252018610386_S01_GE2_105_Dec08_1_4.txt 

> RG <- backgroundCorrect(RG,
method="minimum", offset=1)

> MA <- normalizeWithinArrays(RG,
method="loess")

> MA.avg <- avereps(MA, ID=MA$genes$SystematicName)

> design <- cbind(MU1vsWT1 = c(-1,1,0,0),
MU2vsWT2 = c(0,0,-1,0), MU3vsWT3=c(0,0,0,1) )

> fit <- lmFit(MA.avg, design)

> cont.matrix <- makeContrasts(MUvsWT
= (MU1vsWT1 + MU2vsWT2+MU3vsWT3)/3, levels = design)

> fit2 <- contrasts.fit(fit,
cont.matrix)

> fit2 <- eBayes(fit2)

> topTable(fit2, adjust = "BH")

> output<- topTable(fit2, adjust =
"BH",number=40000)

> write.table(output, file="isp-1
vs. isp-1;ceh-23.txt", sep="\t", quote=FALSE)
my session info is R version 2.14.0 (2011-10-31)Platform: x86_64-pc-mingw32/x64 (64-bit)
locale:[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252    LC_MONETARY=English_United States.1252[4] LC_NUMERIC=C                           LC_TIME=English_United States.1252    
attached base packages:[1] stats     graphics  grDevices utils     datasets  methods   base     
other attached packages:[1] limma_3.10.0
I will be appreciate any feedback.
thanks a lot!
Ella

 		 	   		  
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