[BioC] paired samples and time course experiments in LIMMA

[Lauren Sassoubre]

Hello, 
I have a question about which analysis to do in LIMMA. I did three experiments (three biological replicates) each with one treatment microcosm ("L") and one control microcosm ("D"). For each experiment, I sampled both the treatment and control microcosms at 5 time points (0 hr, 2 hr, 6 hr, 12 hr, 24 hr). With 3 replicate experiments * (1 treatment + 1 control) * 5 time points, I have data from 30 microarrays. 

Based on section 8.6 of the LIMMA manual (which is incredibly helpful thank you!) here is the way I set up my targets table:
FileName	Target
EF731D0.CEL	D.0hr
EF731D2.CEL	D.2hr
EF731D6.CEL	D.6hr
EF731D12.CEL	D.12hr
EF731D24.CEL	D.24hr
EF731L0.CEL	L.0hr
EF731L2.CEL	L.2hr
EF731L6.CEL	L.6hr
EF731L12.CEL	L.12hr
EF731L24.CEL	L.24hr
EF813D0.CEL	D.0hr
EF813D2.CEL	D.2hr
EF813D6.CEL	D.6hr
EF813D12.CEL	D.12hr
EF813D24.CEL	D.24hr
EF813L0.CEL	L.0hr
EF813L2.CEL	L.2hr
EF813L6.CEL	L.6hr
EF813L12.CEL	L.12hr
EF813L24.CEL	L.24hr
EF815D0.CEL	D.0hr
EF815D2.CEL	D.2hr
EF815D6.CEL	D.6hr
EF815D12.CEL	D.12hr
EF815D24.CEL	D.24hr
EF815L0.CEL	L.0hr
EF815L2.CEL	L.2hr
EF815L6.CEL	L.6hr
EF815L12.CEL	L.12hr
EF815L24.CEL	L.24hr

I would like to determine which genes changed over time in the treatment relative to the control at each time point.  Is the following code correct to answer this question??

cont.dif=makeContrasts(Dif2hr=(L.2hr-L.0hr)-(D.2hr-D.0hr), Dif6hr=(L.6hr-L.0hr)-(D.6hr-D.0hr), Dif12hr=(L.12hr-L.0hr)-(D.12hr-D.0hr), levels=design)
fitdif=contrasts.fit(fit, cont.dif)
fitdif=eBayes(fitdif)
topTable(fitdif, genelist=genetext, adjust="BH")

I also would like to determine which genes were differentially expressed between the treatment (L) and control (D) at each time point but I'm not sure which of the following two options to use??

option #1:
contLD.matrix=makeContrasts(L.0hr-D.0hr, L.2hr-D.2hr, L.6hr-D.6hr, L.12hr-D.12hr, L.24hr-D.24hr, levels=design)
fitLDcontmatrix=contrasts.fit(fit, contLD.matrix)
fitLDcontmatrixB=eBayes(fitLDcontmatrix)

OR

option #2: Since each biological replicate experiment starts with the same bacterial culture (half exposed to the treatment while half are kept as a control), I started to think that the treatment and control samples from each experiment are paired. This made me think about doing paired sample tests for each time point. So, following the paired sample section of the LIMMA manual I created a different targets table and did the following code for each time point:

targets:
FileName	Rep	Treatment
EF731D0.CEL	1	D
EF731L0.CEL	1	L
EF813D0.CEL	2	D
EF813L0.CEL	2	L
EF815D0.CEL	3	D
EF815L0.CEL	3	L

code: 
Rep = factor(targets$Rep) # this is like SibShip in the LIMMA example
Treat = factor(targets$Treatment, levels=c("D","L"))
design = model.matrix(~Rep+Treat)
fit0 = lmFit(eset_t0, design)
fit0 = eBayes(fit0)
topTable(fit0, coef="TreatL", genelist=genetext, adjust="BH")

Thanks in advance!
Lauren