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@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" > >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor 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 uiuc.edu