Entering edit mode
Dear Eleonora,
You need to explain what you want to test for before we can advise you
on
the correct contrast.
Also, if your dye-swaps are technical replicates, it appears that your
experiment doesn't have any biological replication. If you've
repeated
the RNA extraction, labelling etc for the dye-swaps then all is well.
If
not, then the experiment is difficult to analyse from the point of
view of
statistical significance.
Best wishes
Gordon
-----------------------------------------------
Associate Professor Gordon K Smyth,
NHMRC Senior Research Fellow,
Bioinformatics Division,
Walter and Eliza Hall Institute of Medical Research,
1G Royal Parade, Parkville, Vic 3052, Australia.
smyth at wehi.edu.au
http://www.wehi.edu.au
http://www.statsci.org/smyth
> Date: Sun, 4 Jul 2010 15:42:13 +0200 (CEST)
> From: "Leleacquario at libero.it" <t>
> To: bioconductor at stat.math.ethz.ch
> Subject: [BioC] Question on Limma technical replicate
>
>
>
> 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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