Dear list I wonder if someone could help me with some conceptual issues regarding limma, contrasts and analysis of an affymetrix study. I have 8 samples (in duplicate) and 3 factors. The factors are O2 level (Norm, Hypo), Radiation (Yes/No) and Treatment (None, DMSO, Drug1, Drug2). The drugs were delivered to the cells using DMSO as a carrier. I am trying to use contrasts to examine the effect of the drug between two O2 levels taking into account the effect of DMSO: TS <- paste(phenoData(rmaDataFiltered)$O2, phenoData(rmaDataFiltered)$Rtx, phenoData(rmaDataFiltered)$Treatment, sep=".") TS <- factor(TS, levels=c("Hypo.No.None", "Hypo.No.DMSO", "Hypo.No.Drug1", "Hypo.No.Drug2", "Hypo.Yes.None", "Hypo.Yes.DMSO", "Hypo.Yes.Drug1", "Hypo.Yes.Drug2", "Norm.No.None", "Norm.No.DMSO", "Norm.No.Drug1", "Norm.No.Drug2", "Norm.Yes.None", "Norm.Yes.DMSO", "Norm.Yes.Drug1", "Norm.Yes.Drug2")) design <- model.matrix(~0+TS) colnames(design) <- levels(TS) fit <- lmFit(rmaDataFiltered, design) cont.matrix <- makeContrasts(HypoDrug1 = (Hypo.No.Drug1-Hypo.No.None)-(Hypo.No.DMSO-Hypo.No.None), NormDrug1 = (Norm.No.Drug1-Norm.No.None)-(Norm.No.DMSO-Norm.No.None), levels=design) For these contrasts my thinking is as follows; I want to examine the effect of the drug versus untreated cells taking into account the effect of DMSO. So first I want to see the effect of DMSO on the untreated cells: (Hypo.No.DMSO-Hypo.No.None) and (Norm.No.DMSO-Norm.No.None) Then I want to remove this effect from the effect of the drug on untreated cells (i.e. create the difference of a difference): HypoDrug1 = (Hypo.No.Drug1-Hypo.No.None)-(Hypo.No.DMSO-Hypo.No.None) (effect of drug - effect of DMSO) and NormDrug1 = (Norm.No.Drug1-Norm.No.None)-(Norm.No.DMSO-Norm.No.None) (effect of drug - effect of DMSO) My question is am i going about this in the right way? Thanks. Iain

Hi Iain, Iain Gallagher wrote: > Dear list > > I wonder if someone could help me with some conceptual issues > regarding limma, contrasts and analysis of an affymetrix study. > > I have 8 samples (in duplicate) and 3 factors. The factors are O2 > level (Norm, Hypo), Radiation (Yes/No) and Treatment (None, DMSO, > Drug1, Drug2). > > The drugs were delivered to the cells using DMSO as a carrier. I am > trying to use contrasts to examine the effect of the drug between two > O2 levels taking into account the effect of DMSO: > > TS <- paste(phenoData(rmaDataFiltered)$O2, > phenoData(rmaDataFiltered)$Rtx, phenoData(rmaDataFiltered)$Treatment, > sep=".") > > TS <- factor(TS, levels=c("Hypo.No.None", "Hypo.No.DMSO", > "Hypo.No.Drug1", "Hypo.No.Drug2", "Hypo.Yes.None", "Hypo.Yes.DMSO", > "Hypo.Yes.Drug1", "Hypo.Yes.Drug2", "Norm.No.None", "Norm.No.DMSO", > "Norm.No.Drug1", "Norm.No.Drug2", "Norm.Yes.None", "Norm.Yes.DMSO", > "Norm.Yes.Drug1", "Norm.Yes.Drug2")) > > design <- model.matrix(~0+TS) colnames(design) <- levels(TS) fit <- > lmFit(rmaDataFiltered, design) > > cont.matrix <- makeContrasts(HypoDrug1 = > (Hypo.No.Drug1-Hypo.No.None)-(Hypo.No.DMSO-Hypo.No.None), NormDrug1 = > (Norm.No.Drug1-Norm.No.None)-(Norm.No.DMSO-Norm.No.None), > levels=design) > > For these contrasts my thinking is as follows; I want to examine the > effect of the drug versus untreated cells taking into account the > effect of DMSO. So first I want to see the effect of DMSO on the > untreated cells: > > (Hypo.No.DMSO-Hypo.No.None) and (Norm.No.DMSO-Norm.No.None) > > Then I want to remove this effect from the effect of the drug on > untreated cells (i.e. create the difference of a difference): > > HypoDrug1 = (Hypo.No.Drug1-Hypo.No.None)-(Hypo.No.DMSO-Hypo.No.None) > (effect of drug - effect of DMSO) Note that algebraically this is the same as Hypo.No.Drug1 - Hypo.No.DMSO > > and > > NormDrug1 = (Norm.No.Drug1-Norm.No.None)-(Norm.No.DMSO-Norm.No.None) > (effect of drug - effect of DMSO) Ditto for this one (Norm.No.Drug1 - Norm.No.DMSO). So you don't need to involve the baseline control samples in these comparisons. > > My question is am i going about this in the right way? Remember that you are just doing algebra here, so it is always possible that a given contrast actually reduces to something much simpler. Best, Jim > > Thanks. > > Iain > > > _______________________________________________ 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 -- James W. MacDonald, M.S. Biostatistician Douglas Lab University of Michigan Department of Human Genetics 5912 Buhl 1241 E. Catherine St. Ann Arbor MI 48109-5618 734-615-7826 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues