Package Limma -contrast matrix differential expression

Question

I am using limma to analyse differential gene expressions. For modeling you need a design and contrast matrix. I just want to know whether anyone has experience with it.

Suppose that expressions are from wild type (WT) and mutants (M) and these are either stimulated (S) or unstimulated (you). For wild type I have 40 expressions values and for mutant 20.

So when I want to know which genes respond differently in mutant compared to wild type, Which formula should I use for the contrast Matrix:

Diff=(M.S-M.U)-(WT.S-M.U) or Diff=(M.S/20-M.U/20)-(WT.S/40-WT.U/40) 

Answer 1

A contrast to determine genes that respond differently in mutant compared to wild type could be:

makeContrasts('WT - M')

where M refers to both the stimulated and unstimulated mutant cells. However, I suspect, you may want something like:

makeContrasts('WT - M.S', 'WT- M.U')

which would highlight changes in the between the WT and either mutant cell.