[BioC] Limma and Dye Flips

[Claus-Dieter Mayer]

Hi Sally,

What you have done (averaging across the dye-flips) is basically ok, as 
the information you loose by doing that only concerns technical 
variability. The more sophisticated way of doing things would be to 
include all arrays, but to change your limma analysis by
a) adding a dye effect (cf. limma tutorial)
b) adding a block effect that informs the analysis about which arrays 
are technical replicates (dye-flips), that analyse the same biological 
samples (again see the limma tutorial for details).

In addition to your treatment effect this analysis will give you 
information about gene-specific dye-effects and the correlation between 
the two dye-flips. Both of these might not be relevant to the biological 
question of interest, but be helpful from a quality control perspective.
If you average the dye-flips you eliminate  the gene-specific dye-effect 
(which is good), but you also loose the information about it. Equally 
you give up the information about the technical array to array (+dye 
flip)-variation. The fold-changes you estimate for your biological 
effect of interest will be identical for both approaches but p-values 
etc will differ slightly. From the point of statistical power both 
approaches should be similar as far as I can see (and others may correct 
me if I overlooked any subtle points here).

Best Wishes

Claus



Sally wrote:
> My question is about handling dye flips in Limma.
>
> In my experiment I did a dye flip for each comparison.  When I finished the preprocessing I averaged the M values of the two dye flip arrays.Now I am fitting a linear model.  Should I have averaged the dye flips before, or should the dye flips be incorporated in the linear model?
>
> Sally Goldes
> 	[[alternative HTML version deleted]]
>
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-- 
***********************************************************************************
 Dr Claus-D. Mayer                    | http://www.bioss.ac.uk
 Biomathematics & Statistics Scotland | email: claus at bioss.ac.uk
 Rowett Research Institute            | Telephone: +44 (0) 1224 716652
 Aberdeen AB21 9SB, Scotland, UK.     | Fax: +44 (0) 1224 715349