# [BioC] Limma time course with two-color microarrays

Gordon K Smyth smyth at wehi.EDU.AU
Thu Mar 15 12:17:59 CET 2007

```> Date: Wed, 14 Mar 2007 10:19:32 -0800 (PST)
> From: "Sergio Barberan" <barberan at biology.ucsc.edu>
> Subject: [BioC] Limma time course with two-color microarrays
> To: bioconductor at stat.math.ethz.ch
>
> Dear list,
>
> It might be a really easy problem but I can not figure out how to do it.
>
> I'm trying to analyze a set of hybs of a time course experiment, where I
> used a loop design. I have six samples and no reference, I compare
> (1-2,2-3,3-4,4-5,5-6,6-1); each with dye swaps so I have 12 slides.
>
> By selecting sample 1 as my reference in modelMatrix, I get the following
> design matrix:
>
>  2 3 4 5 6
> 1      0 -1  1  0  0
> 2      0  0 -1  1  0
> 3      0  0  0 -1  1
> 4      1  0  0  0 -1
> 5      0  1  0  0  0
> 6      0  0  0  1 -1
> 7      0  1 -1  0  0
> 8     -1  0  0  0  1
> 9      1  0  0  0  0
> 10     0  0  1 -1  0
> 11     0 -1  0  0  0
> 12    -1  0  0  0  0
>
> My question is: How to use contrastMatrix to obtain the following
> comparisons: 1-2, 2-3, 3-4, 4-5, 5-6 and 6-1?

You just have to remember that each column of your design matrix is already a contrast vs 1.  So
you need

cont.matrix <- makeContrasts(-2,2-3,3-4,4-5,5-6,6,levels=design)

HOWEVER, to make this work the column names of the your design matrix must start with a letter,
not a number.  E.g., you need "Time2", "Time3" etc instead of "2", "3" etc.

> I know there is some information about Limma and time course experiments
> but that example uses one-color arrays so I'm confused about how to apply
> that to my experimental design.

The two colour is the same as the one-colour from this point.

Best wishes
Gordon

> Thanks in advance for any help,
> Sergio

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