[BioC] Help needed on limma and differentially expressed genes

[Stanley Ng]

Hi Everyone, 

Apology, earlier two emails sent out via gmail and outlook messed up the
alignment. Even I looking at them, confused me. So finally this last post
got a response. 

Using Jenny's suggestion, I had similar idea that Wt0 samples were
unneccessary. But try to explain that to a biologist, is much tougher as Wt0
is deemed the baseline. 

Couple of questions from my part:
A) Does E8 - Wt0 really exclude the effect of the empty plasmid ? I am
impressed by the limma user guide that the "-" sign means solely comparison
in the log2 scale and would mean a fold change in the raw intensity.  
B) the pooled RNA were absent in the contrast matrix, can I safely set
ref="Pooled RNA" in the design matrix  

More help please. 

Thanks finally.
Stanley

-----Original Message-----
From: Jenny Drnevich [mailto:drnevich at uiuc.edu] 
Sent: Tuesday, April 08, 2008 23:09
To: Ng Stanley; bioconductor at stat.math.ethz.ch
Subject: Re: [BioC] Help needed on limma and differentially expressed genes

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.
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