[BioC] Limma-design matrix for technical replication

[Jenny Drnevich]

Hi Katerina,

There is an example of how to deal with both biological and technical 
replicates in the limma vignette, section 8.2. There is even an 
example for dye-swaps, which I believe fits your design exactly. To 
get the limma vignette:

 > limmaUsersGuide()

Good luck,
Jenny

At 10:06 AM 8/11/2008, =?iso-8859-2?Q?Kate=F8ina_Kepkov=E1?= wrote:
>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
>cells.
>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 =
>design)
>fit2 <- contrasts.fit(fit, cont.matrix)
>fit2 <- eBayes(fit2)
>
>
>Sorry if I am asking something obvious and thank you in advance for your
>help.
>
>Best regards,
>Katerina
>
>---------------------------------------------------------------------
>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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Jenny Drnevich, Ph.D.

Functional Genomics Bioinformatics Specialist
W.M. Keck Center for Comparative and Functional Genomics
Roy J. Carver Biotechnology Center
University of Illinois, Urbana-Champaign

330 ERML
1201 W. Gregory Dr.
Urbana, IL 61801
USA

ph: 217-244-7355
fax: 217-265-5066
e-mail: drnevich at illinois.edu