> Date: Thu, 06 Oct 2005 12:40:32 +0300 > From: "Ron Ophir" <ron.ophir at="" weizmann.ac.il=""> > Subject: [BioC] [limma] Factorial refernce design > To: "<bioconcutor" <bioconductor="" at="" stat.math.ethz.ch=""> > > Hi, > I work with dual channel technology and I would like to use limma for > two way analysis. My experiment is as follow: > FileName Cy3 Cy5 > GL_251322310100_S02_.gpr WT3h NI3h > GL_251322310101_S02_.gpr WT3h NI3h > GL_251322310103_S02_.gpr Mut3h NI3h > GL_251322310104_S02_.gpr Mut3h NI3h > GL_251322310107_S02_.gpr WT18h NI3h > GL_251322310136_S02_.gpr WT18h NI3h > GL_251322310108_S02_.gpr Mut18h NI3h > GL_251322310368_S02_.gpr Mut18h NI3h > where I have two factors genotype: WT and Mut and Time: early and late > and NI3h is the reference. > Therefore it seems to be natural that my design matrix would be as > WT3h Mut3h WT18h Mut18h > GL_251322310100_S01 -1 0 0 0 > GL_251322310101_S01 -1 0 0 0 > GL_251322310103_S01 0 -1 0 0 > GL_251322310104_S01 0 -1 0 0 > GL_251322310107_S01 0 0 -1 0 > GL_251322310136_S01 0 0 -1 0 > GL_251322310108_S01 0 0 0 -1 > GL_251322310368_S01 0 0 0 -1 > > then run fit<-lmFit(Marray,design) > > I want to answer the following questions: > What is the effect of genotype on genes' expression? > What is the effect of time on genes' expression? > What is the synergistic effect of time and genotype? > > Since the most intuitive for me is to use the sum to zero > parameterization method I created such contrast matrix > > Genotype.WTvsMut Time.EarlyvsLate Int > WT3h 1 -1 1 > Mut3h -1 -1 -1 > WT18h 1 1 -1 > Mut18h -1 1 1 > > then run contrasts<-contrasts.fit(fit,cotrasts.matrix) > > However from the example in chapter 14 in Limma users guide it seems > that I had to run the my contrats matrix with lmFit function. I can't guess what you mean, or what difficulty you are imagining. The section on factorial designs in the users guide is exactly equivalent to what you have given above. Gordon > I allowed myself to use the example in chapter 14 since reference > design equivalent to affymetrix experiment after parametrizing it as > mentioned above with my design matrix and fit it. > So is it right the way I did it although in the example for factorial > design my design of contrasts matrix is run with lmFit function? > Is OK to run it without the intercept WT=c(1,1,1,1) if I'm not > interesting in it? > > Thanks in advance, > Ron