Question: limma: writing down the matrix in an almost (?) factorial design experiment
0
9.8 years ago by
Massimo Pinto390
Massimo Pinto390 wrote:
microarray dose • 642 views
modified 9.7 years ago • written 9.8 years ago by Massimo Pinto390
Answer: limma: writing down the matrix in an almost (?) factorial design experiment
0
9.7 years ago by
Massimo Pinto390
Massimo Pinto390 wrote:
I thought I should add here how the design matrix was created. Probably, if I understand the language correctly, this is a mixed factorial/nested design with three two-level factors: Dose <- factor(targets$Dose, levels=c("Cn", "1Gy")) # It's very important that you define the levels now, with their order, as otherwise he will do it for you and this may not be the way you want it. Lab <- factor(targets$Lab, levels=c("ISS", "LNGS")) Ageing <- factor(targets\$Ageing, levels=c("t0", "6mo")) disegno.3 <- model.matrix(~Dose + Ageing + Dose:Ageing + Ageing:Lab + Dose:Ageing:Lab +1) Yours, Massimo Massimo Pinto Post Doctoral Research Fellow Enrico Fermi Centre and Italian Public Health Research Institute (ISS) Rome, Italy http://claimid.com/massimopinto On Thu, Feb 11, 2010 at 11:07, Massimo Pinto <pintarello at="" gmail.com=""> wrote: > Dear all, > > I am writing in relation to some doubts that I have on the > implementation of a design matrix in a factorial design experiment. I > have tried to follow, religiously, the recommendations as at paragraph > 23.10 of the R&Bioconductor monography, but have come up with several > afterthoughts. > > Here is my experiment. Together with my colleagues, we have taken a human cell > culture, the human lymphoblastoid line TK6, and split it in two > laboratories with > different environmental conditions. Specifically, environmental > background ionizing radiation differs across the labs by a factor of > 80. We are asking whether continuous cell culture under reduced > environmental background radiation renders cells more or less tolerant > to acute exposure to genotoxic or cytotoxic agents, a line of research > that the lab in which I am working has been busy for several years. > These two labs, and here is the first factor, are called "ISS" and > "LNGS" in what follows. We have > isolated total RNA samples (all instances contain 4 biological > replicates) from these two cell cultures after they have been > maintained in each laboratory for six months (corresponding to hundreds of cell > generations) since we hypothesized that it took several cell > generations before deviations at the level of the transcriptome may > reveal. Therefore, a second variable (factor) is time, and this has > the values "0" and "6 months". > As you imagine, we have a determination on RNA extracted from the "0" > time sample, which is the 'mother' cell > culture of both "ISS" and "LNGS" (6 months) cultures. > Additionally, to reveal any subtle differences between the two aged > cultures, we have exposed them to an acute dose of ionizing radiation, > which acts as a stressor. Our third variable/factor, therefore, is radiation > Dose, which takes the values "0" and "1 Gy". Accordingly, we have an > RNA extraction and a microarray determination at time = "0" and Dose = > "1Gy", to compare with responses ?to radiation after six months in > each laboratory. > Following the BioC Monography, I have applied the model.matrix() > function obtaining > a design matrix with 8 columns. Two such columns, howewer, have no > sense to this particular experiment, since the variable Laboratory > ("ISS", "LNGS") > has no meaning unless 6 months have elapsed, since their source is the > same culture, the "0" culture from which the experiment started. > Accordingly, I deleted - by hand - those two columns and obtained the > attached design matrix. > Do you think that this matrix is correct? We have applied it for some > time in our analysis and felt sure it was, but we have come across to > some odd results with respect to all analyses of 1Gy-irradiated > samples and we are now re-evaluating this matrix. > > One anchoring point in our reasoning is what follows. The samples > exposed to 1Gy after six months in culture in both labs (ISS, LNGS) > are not directly derived from samples exposed to the same 1Gy dose at > time "0". Those would be survivors to the 1Gy dose delivered 6 months > before, which is not a question being addressed in this experiment > (though a reasonable question). Instead, samples exposed to 1Gy are > always derived from > their unirradiated counterparts at the same time and in the same > laboratory. For example, the LNGS, 6 months, 1Gy-irradiated sample is obtained > from exposure to 1Gy of the sample maintained for six months at LNGS, > again, not by maintaining for six months at LNGS the sample that had been > exposed to 1Gy dose at time "0". > > I want to thank you very much in advance for the patience that you > have put in reading all down here and I hope to read any suggestions. > > yours, > > Massimo P. >