On 8/16/06 4:15 PM, "Jailwala, Parthav" <pjailwal at="" mcw.edu=""> wrote:
> Hi,
>
>
>
> I am new to this usergroup and also a new user of Limma. I have a
> question related to the targets.txt file which is input to Limma for
> Imagene generated intensity data.
>
> For each slide, I have a Cy3 file and a Cy5 file. In the targets
file, I
> specify the respective files under the columns 'FileNameCy3' and
> 'FileNameCy5'. As I understand from the limma users' guide, the
> FileNameCy3 files correspond to the 'Green ' channel and the
FileNameCy5
> files correspond to the 'Red' channel.
>
> My question is, while calculating the M in the MA algorithm, is the
> ratio always going to be log2(Red-FileNameCy5 / Green-FileNameCy3) ?
>
> I particularly wish to know this, as we have a dye-flip design,
where
> consequtive slides have the dye-assignments reversed for Stimulated
and
> unstimulated samples. So in the resulting output file after
> normalization (if the ratios are always Cy5/Cy3), I should be
actually
> taking the negative of the log ratios for those dye-flipped slides
to
> arrive at the 'Stimulated/Unstimulated' ratios for comparison.
In limma, the effect of dye is part of the "analysis", not the
normalization. Just leave the red/green columns as they are. So,
yes, some
of your ratios will be upside-down from what you want. However, when
you
set up your design matrix for analysis, the dye swap nature can be
accounted
for. There are a number of examples in the documentation that use
two-color
arrays with dye-swaps; you may want to check those out to convince
yourself
that this is an OK way to go.
Sean
