[BioC] two way anova with interaction effect : limma package

[John Antony Gaspar]

Dear Bioc,

I have 5 groups of samples with their WT type, Diff type and Diff & pure 
type. To make it clear
aES,aDiff,aDiff_pure,  bES,bDiff,bDiff_pure, cES,aDiff,cDiff_pure, 
dES,dDiff,dDiff_pure, eES,eDiff,eDiff_pure.

I wish to do two way anova (differentiation and pure_treatment) calculation.
Therefore I just try with the following code with limma package:
-----
Diff<-factor(c(1,1,1,2,2,2,2,2,2,1,1,1,2,2,2,2,2,2,1,1,1,2,2,2,2,2,2,1,1,1,2,2,2,2,2,2,1,1,1,2,2,2,2,2,2))

Cond<-factor(c(1,1,1,1,1,1,2,2,2,1,1,1,1,1,1,2,2,2,1,1,1,1,1,1,2,2,2,1,1,1,1,1,1,2,2,2,1,1,1,1,1,1,2,2,2))

design<-model.matrix(~Diff+Cond)

fit<-lmFit(limma_data,design)

fit<-eBayes(fit)

limma_deg_result<-topTableF(fit,number=45101,genelist=fit$genes,adjust.method="BH")
limma_deg_result <- limma_deg_result[limma_deg_result$adj.P.Val<=0.05,]
write.table(limma_deg_result, "DEgenes.txt", quote=FALSE, 
row.names=FALSE, sep="\t")

-------

Of course, I ended up with a DEgenes.txt table.
However my worry is to know whether am I right in setting design.
and another thing is I wish to have the interaction effect of Diff and Cond.
In that case in what way I have to set model in design.

I may be a fool to do this with insufficient knowledge I have to have 
the interaction effect by setting design as it follows
design<-model.matrix(~Diff*Cond)

I am ended up with the following problem:

/Coefficients not estimable: Diff2:Cond2
Warning message:
Partial NA coefficients for 45101 probe(s)/

Please correct me where I do mistake.


Thanking you in advance,
antony