Hello guys,

I currently have the following design: (Knockout=TAm and WT-Veh) in a time course (0,3,5 and 7 days)

> design
   fTam.0d fTam.1d fTam.3d fTam.5d fTam.7d fVeh.0d fVeh.1d fVeh.3d fVeh.5d fVeh.7d
1        1       0       0       0       0       0       0       0       0       0
2        1       0       0       0       0       0       0       0       0       0
3        0       1       0       0       0       0       0       0       0       0
4        0       1       0       0       0       0       0       0       0       0
5        0       0       1       0       0       0       0       0       0       0
6        0       0       1       0       0       0       0       0       0       0
7        0       0       0       1       0       0       0       0       0       0
8        0       0       0       1       0       0       0       0       0       0
9        0       0       0       0       1       0       0       0       0       0
10       0       0       0       0       1       0       0       0       0       0
11       0       0       0       0       0       1       0       0       0       0
12       0       0       0       0       0       1       0       0       0       0
13       0       0       0       0       0       0       1       0       0       0
14       0       0       0       0       0       0       1       0       0       0
15       0       0       0       0       0       0       0       1       0       0
16       0       0       0       0       0       0       0       1       0       0
17       0       0       0       0       0       0       0       0       1       0
18       0       0       0       0       0       0       0       0       1       0
19       0       0       0       0       0       0       0       0       0       1
20       0       0       0       0       0       0       0       0       0       1

I want to compare within the genotype as well as time-course. I've been following the Limma section 9.6.1 (Time course with replicates) and my strategy looks like:

 > colnames(design)
 [1] "Tam.0d" "Tam.1d" "Tam.3d" "Tam.5d" "Tam.7d" "Veh.0d" "Veh.1d" "Veh.3d" "Veh.5d" "Veh.7d"

> fit<- lmFit(dat, design)

> cont.Tam<- makeContrasts("Tam.1d-Tam.0d", "Tam.3d-Tam.0d", "Tam.5d-Tam.0d", "Tam.7d-Tam.0d", levels = design)

> fit2<- contrasts.fit(fit, cont.Tam)

> fit2<- eBayes(fit2)

I would do the same for the WT as in the manual. My question is, am I doing this right, to have an Anova-like test? If so, how can I get the differences? Based on I saw it would be:

>topTable(fit2, coef = 1, number=Inf, adjust.method = "BH")

I just want to make sure if I'm doing the proper design and comparisons and how to get what is actually changing.

Your analysis seems fine from the code you've shown. So far you have obtained a list of differentially expressed genes for KO at 1 day vs KO at 0 days. You can perform any other comparison you want in the same way.