A subset of Agilent arrays I'm working a set of arrays in which a subset
were were not dye swapped.  I would like to use all of the array data if
possible/ 

 

Exp 1: The dye-swapped batch compared 10 individuals before and after
treatment, so I have red and green for each under each condition. 

 

Exp 2: The non-dye-swapped subset compared pooled RNA from the group
(cy3) to RNA from each the 10 individuals (cy5), both with no treatment.

 

Both sets of arrays were prepared and scanned within one week of each
other.

 

I would have thought that the cy3 for the pool from Exp. 1 would be very
close to the cy3 values of individual data from Exp 1.   Instead the
green intensities are much stronger from the pool than those from the
individuals in Exp. 1 . I am also finding many differentially expressed
genes when analyzing Exp 2 even after normalization (loess). 

 

Shouldn't the log cy5/cy3 ratios be close to 1  when the pool is made up
of the same individuals?  I believe that the problem is that the green
dye intensities are the problem.

 

Is there a way to adjust for this? 

 

Thanks,

 

Kathryn

 

 


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