As I have noted elsewhere in the list, it is often easier to do the analysis using Single Channel analysis, particularly if the only blocking factor is the array. The 2-way ANOVA is certainly more easily set up this way, although depending on the design, a 2x2 can be done using 2-channel analysis as long as one is very careful about setting up the contrasts. (Sorry, I am not up for setting this up for anyone at present.) --Naomi At 01:31 PM 7/13/2009, Robert Castelo wrote: >hi Giusy, > >so then I understand that the only way to go with two-color 2x2 >factorial design without a common reference RNA source is to treat each >combination of factors as an independent experiment, right? > >(i initially understood from the last emails that there was an >alternative by using the parameters argument to the modelMatrix() >function, but i understand now this alternative cannot be employed) > >robert. > >On Mon, 2009-07-13 at 11:23 -0400, Giusy Della Gatta wrote: > > Hi Robert, > > I saw the example used in chapter 11.5. The problem is that > > I don't have any common reference that I can use to normalize > > the data, while in the chapter they are a comparing everything against > > the "Pool". > > > > > > G > > > > > > > > > > -----Original Message----- > > From: Robert Castelo [mailto:robert.castelo at upf.edu] > > Sent: Monday, July 13, 2009 6:00 AM > > To: James W. MacDonald > > Cc: Giusy Della Gatta; bioconductor at stat.math.ethz.ch > > Subject: Re: [BioC] design a modelMatrix with no common references > > > > James, Guisy, > > > > if you let me make a question about what you're discussing. > > > > why do you say that the two-color 2x2 factorial design without a common > > reference RNA source is "a lot of work" ?? > > > > (i was actually wondering how would be the 11.5 example of the Limma > > user's guide without a common RNA source) > > > > thanks! > > robert. > > > > On Fri, 2009-07-10 at 10:14 -0400, James W. MacDonald wrote: > > > Hi Guisy, > > > > > > You really have two different experiments here, so I don't know if limma > > > is going to want to do things automatically for you without warnings or > > > incorrect model matrices. However, I think you want to use the > > > parameters argument to modelMatrix() rather than the ref argument (since > > > you have two different reference samples). > > > > > > > targets <- matrix(paste(rep(c("Myc","Rag"), each=4), > > > rep(c("CD3","PBS"), each=2, times=3)[2:9], sep = "_"), byrow=T, ncol=2) > > > > targets > > > [,1] [,2] > > > [1,] "Myc_CD3" "Myc_PBS" > > > [2,] "Myc_PBS" "Myc_CD3" > > > [3,] "Rag_CD3" "Rag_PBS" > > > [4,] "Rag_PBS" "Rag_CD3" > > > > colnames(targets) <- c("Cy3","Cy5") > > > > rownames(targets) <- paste("Array", 1:4) > > > > targets > > > Cy3 Cy5 > > > Array 1 "Myc_CD3" "Myc_PBS" > > > Array 2 "Myc_PBS" "Myc_CD3" > > > Array 3 "Rag_CD3" "Rag_PBS" > > > Array 4 "Rag_PBS" "Rag_CD3" > > > > parameters <- cbind(First=c(-1,1,0,0), Second=c(0,0,-1,1)) > > > > rownames(parameters) <- c("Myc_PBS","Myc_CD3","Rag_PBS","Rag_CD3") > > > > parameters > > > First Second > > > Myc_PBS -1 0 > > > Myc_CD3 1 0 > > > Rag_PBS 0 -1 > > > Rag_CD3 0 1 > > > > modelMatrix(targets, parameters) > > > Found unique target names: > > > Myc_CD3 Myc_PBS Rag_CD3 Rag_PBS > > > First Second > > > Array 1 -1 0 > > > Array 2 1 0 > > > Array 3 0 -1 > > > Array 4 0 1 > > > Warning message: > > > In modelMatrix(targets, parameters) : > > > number of parameters should be one less than number of targets > > > > > > But that seems like a lot of work, as the parameters matrix is exactly > > > the model matrix you want. > > > > > > Best > > > > > > Giusy Della Gatta wrote: > > > > Hi everybody, > > > > > > > > I have Agilent two colors expression arrays in which have > been analyzed > > > > two KO mice samples (myc-/- and Rag -/-) treated with CD3 and with PBS. > > > > I have a total of 4 arrays composed as follows: > > > > Sample Cy3 Cy5 > > > > 1. Myc24CD3 Myc_CD3 Myc_PBS (Swap) > > > > 2. Myc24PBS Myc_PBS Myc_CD3 > > > > 3. Rag24CD3 Rag_CD3 Rag_PBS (Swap) > > > > 4. Rag24PBS Rag_PBS Rag_CD3 > > > > > > > > After the normalization I don't know > > > > how to proceed for the construction of the model matrix. > > > > > > > > By using the suggestions of the "Direct Two Color Designs" > example (chapter 7.4 LIMMA guide) > > > > I did: > > > > > > > > > > > >> targets > > > > FileName Cy3 Cy5 Collection_time > > > > 1 3_Myc24CD3gr_Myc24PBSre Myc_CD3 Myc_PBS 24h > > > > 2 9_Myc24PBSgr_Myc24CD3re Myc_PBS Myc_CD3 24h > > > > 3 5_Rag24CD3gr_Rag24PBSre Rag_CD3 Rag_PBS 24h > > > > 4 4_Rag24PBSgr_Rag24CD3re Rag_PBS Rag_CD3 24h > > > > > > > >> designmyc= modelMatrix(targets, ref="Myc_PBS") > > > > Found unique target names: > > > > Myc_CD3 Myc_PBS Rag_CD3 Rag_PBS > > > > > > > >> designmyc > > > > Myc_CD3 Rag_CD3 Rag_PBS > > > > [1,] -1 0 0 > > > > [2,] 1 0 0 > > > > [3,] 0 -1 1 > > > > [4,] 0 1 -1 > > > > > > > >> fit = lmFit(MA.Rq, designmyc) > > > > Coefficients not estimable: Rag_PBS > > > > Warning message: > > > > Partial NA coefficients for 45018 probe(s) > > > > > > > > > > > > But at this point I calculated just the ratios of Myc_CD3/Myc_PBS > > > > and Rag_Myc/Myc_PBS (I am not really interested in this last one!). > > > > How can I specify in the model matrix design that I need two > different references > > > > to calculate the following logratios: Myc_CD3/Myc_PBS, > Rag_Myc/Rag_PBS? > > > > > > > > > > > > Thank you in advance! > > > > Giusy > > > > > > > > > > > > _______________________________________________ > > > > 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 > > > > > > > > >_______________________________________________ >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 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