[BioC] limma Dye Swap question

[Ramon Diaz-Uriarte]

Dear Mike,

There have been several threads about this, or similar, issues in the BioC 
list. Two I recall were on april 2004 and august 2003, but I think this issue 
has come up several other times. You can download the BioC archives and 
search for "technical replicates" and "dye swap".

Best,

R.


P.S. A more general question, though, is why you use tech. reps. If 
availability of biological material is not a problem, this might not be the 
best design for a fixed number of arrays (this issue is discussed in the 
review papers by Speed and Yang, Churchill, and the book by Simon et al.).



On Wednesday 15 September 2004 21:41, Mike Schaffer wrote:
> I had a question about how limma handles dye swap experiments.
>
> The way we typically run these experiments is to label an aliquot of a
> test set of (amplified) aRNA with Cy3 and another aliquot of the same
> aRNA with Cy5.  We do the same with the control aRNA and hyb opposite
> dyes on two slides (i.e. slide 1: test Cy5/control Cy 3, slide 2: test
> Cy3/control Cy 5).  In the past we would loess normalize the slides,
> calculate an average log2 ratio for the set, and use 1 number to
> represent the experimental M value from the two slides.
>
> Q: Is there any consideration in limma given to the fact that these two
> slides represent the same original RNA sample and therefore (partially)
> represent a technical replicate?
>
> It appears that limma lumps all of the slides together regardless of
> which slides are supposed to be paired with others.  It seems to me
> that these pairs of slides must be considered independently from other
> biological dye-swap replicates.  Can anyone shed some light on how
> limma handles this?
>
> Thanks in advance.
>
> Mike Schaffer
> Ph.D. Candidate
> Bioinformatics Program
> Boston University
>
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-- 
Ramón Díaz-Uriarte
Bioinformatics Unit
Centro Nacional de Investigaciones Oncológicas (CNIO)
(Spanish National Cancer Center)
Melchor Fernández Almagro, 3
28029 Madrid (Spain)
Fax: +-34-91-224-6972
Phone: +-34-91-224-6900

http://ligarto.org/rdiaz
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