[BioC] Help needed on limma and differentially expressed genes

[Jenny Drnevich]

Hi Stanley,

In general, posting the same question 3 times to the list is LESS 
likely to get it answered; however, your 3rd posting was finally 
clear enough to understand. The design matrix you want to use is for 
a common reference design (7.3 in limma vignette), which will give 
you one column for each of your conditions: wild type 0h (Wt0), wild 
type empty plasmid 8 hr (E8), wild type P53 plasmid 8 hr (P8), E16 
and P16. Then it is just a matter of setting up your contrasts 
"correctly". Based on my interpretation of what you want, I think 
that the Wt0 samples were unnecessary! My logic:

a) You want the differently expressed genes induced by P8 compared to 
Wt0:    P8 - Wt0

b) But you also want to exclude the effect of the empty plasmid, 
which is: E8 - Wt0

c) Putting the two together: (P8 - Wt0) - (E8 - Wt0)  =  P8 - E8

d) Likewise, to get the effect at 16h compared to 0h you would use: P16 - E16

e) The difference between 16h and 8 hr would be: (P16 - E16) - (P8 - E8)


HTH,
Jenny

At 01:50 AM 4/8/2008, Ng Stanley wrote:
>Hi,
>
>The design model is attached to this email.
>
>I am using limma to analyze a set of microarray, but face with the
>difficulty on the design matrix and contrast matrix even after reading the
>limma userguide carefully. Basically, I need to exclude the effects of empty
>plasmid transfected into the cells. Then identify
>
>1) differentially expressed genes at 8h and 16h induced by the
>over-expression of P53 with respect to 0h,
>2) those genes that are differentially expressed from 8h to 16h  induced by
>the over-expression of P53.
>
>The experiment assumes that Wild type at 8h and 16h are the same as at 0h,
>due to cost limitations.
>
>Please help.
>
>Thanks
>Stanley
>
>Content-Type: application/pdf; name="Design Model.pdf"
>X-Attachment-Id: f_fes3u99s0
>Content-Disposition: attachment; filename="Design Model.pdf"
>
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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

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e-mail: drnevich at uiuc.edu