Hi, everyone, I am working on a dual-channel cDNA microarray project which includes 32 slides as a loop design. It is a 2x2x2x2 factorial experimental design that combines 16 treatments. When I was trying to use modelMatrix to design the matrix, it always asked me to input a reference. However, in my experiment, I don't really have one as a reference. I tried to assign a random treatment as reference but that way I couldn't make contrasts of the reference with the other treatments. How should I set this argument? Is there a different way that allows me not to assign a reference? Thanks. Wei -- Wei Xu Aquatic Animal Health Laboratory Pathobiology and Diagnostic Investigation College of Veterinary Medicine Michigan State University East Lansing, MI, 48824 Tel: 1-517-432-8258 [[alternative HTML version deleted]]

Since you are using a loop design, you should have 2 replicates per factor combination. In that case, you should be able to fit up to 4-way interactions. --Naomi At 01:59 PM 1/19/2009, Wei Xu wrote: >Thanks, Gordon and Naomi. >One of the examples in limma manual talks about 2x2 factorial >experiments of a two-channel array. It estimates the interaction in >the makeContrasts. > > cont.matrix <- makeContrasts( >+ WT11.MT11=P11mt-P11wt, >+ WT21.MT21=P21mt-P21wt, >+ WT11.WT21=P21wt-P11wt, >+ MT11.MT21=P21mt-P11mt, >+ Int=(P21mt-P11mt)-(P21wt-P11wt), >+ levels=design) >Since I got more factors (2x2x2x2), can I make the interactions with >more factors (i.e. two-way, three-way, and four-way interactions)? > >Wei > >On Fri, Jan 16, 2009 at 11:10 PM, Naomi Altman ><<mailto:naomi@stat.psu.edu>naomi@stat.psu.edu> wrote: >I think that Wei Xu is referring to the fact that one ANOVA is not >an identifiable model without a constraint - hence one either has to >construct the model matrix with an indicator variable, or with the >factor effects. > >In limma, this does not matter, because limma does the overall >F-test and then allows you to construct as many contrasts as are reasonable. > >I always use single channel analysis for loop designs, which makes >it easier to set up the main effects and interactions. > >This was a kind of "statsy" comment on my part, but I hope that >those who have taken a course including factorial designs can follow. > >--Naomi > > > >At 10:33 PM 1/16/2009, Gordon K Smyth wrote: > >Date: Thu, 15 Jan 2009 16:12:08 -0500 >From: Wei Xu <<mailto:xuwei3@msu.edu>xuwei3@msu.edu> >Subject: [BioC] reference argument in limma factorial design >To: <mailto:bioconductor@stat.math.ethz.ch>bioconductor@stat.math.ethz.ch >Content-Type: text/plain > >Hi, everyone, > >I am working on a dual-channel cDNA microarray project which includes 32 >slides as a loop design. It is a 2x2x2x2 factorial experimental design that >combines 16 treatments. When I was trying to use modelMatrix to design the >matrix, it always asked me to input a reference. However, in my experiment, >I don't really have one as a reference. I tried to assign a random treatment >as reference but that way I couldn't make contrasts of the reference with >the other treatments. > > >Yes, you can make contrasts between the reference and other >treatments, in fact that is what the coefficients now mean. Please >refer to the examples on common reference designs in the limma >User's Guide (for example Section 7.3), or past advice given on this list. > >Best wisehs >Gordon > >How should I set this argument? Is there a different >way that allows me not to assign a reference? Thanks. > >Wei > >-- >Wei Xu >Aquatic Animal Health Laboratory >Pathobiology and Diagnostic Investigation >College of Veterinary Medicine >Michigan State University >East Lansing, MI, 48824 >Tel: 1-517-432-8258 > > >_______________________________________________ >Bioconductor mailing list ><mailto:bioconductor@stat.math.ethz.ch>Bioconductor@stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: ><http: news.gmane.org="" gmane.science.biology.informatics.conductor="">ht tp://news.gmane.org/gmane.science.biology.informatics.conductor > > >Naomi S. Altman 814-865-3791 (voice) >Associate Professor >Dept. of Statistics 814-863-7114 (fax) >Penn State University 814-865-1348 (Statistics) >University Park, PA 16802-2111 > > > > > >-- >Wei Xu >Aquatic Animal Health Laboratory >Pathobiology and Diagnostic Investigation >College of Veterinary Medicine >Michigan State University >East Lansing, MI, 48824 >Tel: 1-517-432-8258 Naomi S. Altman 814-865-3791 (voice) Associate Professor Dept. of Statistics 814-863-7114 (fax) Penn State University 814-865-1348 (Statistics) University Park, PA 16802-2111 [[alternative HTML version deleted]]