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Mike Schaffer
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@mike-schaffer-424
Last seen 10.3 years ago
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