[BioC] limma: writing down the matrix in an almost (?) factorial design experiment

[Massimo Pinto]

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.
>