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.

I am afraid I might not have stated the problem clearly...have tried the
best. I would really appreciate if one of the expert users can give me
some clues to this.

 

Thanks

Parthav Jailwala

Analyst

McGee center

Medical College of Wisconsin

 

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It is worse to be insincere than to be in error! 

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