[BioC] Limma-design matrix for technical replication

Kateřina Kepková kepkova at iapg.cas.cz
Mon Aug 11 17:06:02 CEST 2008

Dear all,
As a complete newbie to microarrays, I am trying to analyze experiment with
following design: Two samples (differentiated versus undifferentiated cells)
were compared directly on two-color oligo array, with 3 biological
replicates (different cell sources) and 4 technical replicates (arrays) per
biological replicate (12 arrays altogether). In every set of technical
replicates two arrays are dye-swap. I am not sure how to handle the
technical and biological replication when trying to fit linear model. We are
interested just in overall comparison differentiated versus undifferentiated
I have arrived to following setup:
Targets file is:
SlideNumber	FileName	Cy3	Cy5
1	1.gpr	N1	D1
2	2.gpr	N1	D1
3	3.gpr	D1	N1
4	4.gpr	D1	N1
5	5.gpr	N2	D2
6	6.gpr	N2	D2
7	7.gpr	D2	N2
8	8.gpr	D2	N2
9	9.gpr	N3	D3
10	10.gpr	N3	D3
11	11.gpr	D3	N3
12	12.gpr	D3	N3

Where N means undifferentiated and D differentiated cells and 1-3 are
biological replicates.

Is the following design correct one? Or is there a better way to obtain
relevant information?
Is this extensible for more/less biological replicates?

design <- cbind(D1vsN1 = c(1,1,-1,-1,0,0,0,0,0,0,0,0), D2vsN2 =
c(0,0,0,0,1,1,-1,-1,0,0,0,0), D3vsN3 = c(0,0,0,0,0,0,0,0,1,1,-1,-1))
fit <- lmFit(MA, design)
cont.matrix <- makeContrasts(DvsN = (D1vsN1 + D2vsN2 + D3vsN3)/3, levels =
fit2 <- contrasts.fit(fit, cont.matrix)
fit2 <- eBayes(fit2)

Sorry if I am asking something obvious and thank you in advance for your

Best regards,

Katerina Kepkova
Laboratory of developmental biology 
Department of Reproductive and Developmental Biology 
Institute of Animal Physiology and Genetics of the AS  CR, v.v.i.
Rumburska 89, Libechov 277 21
Czech Republic
tel:     +420 315 639 534
fax:     +420 315 639 510
e-mail: kepkova at iapg.cas.cz

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