Package Limma -contrast matrix differential expression

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 unstimula开发者_如何转开发ted (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) 

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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.