I have encountered a problem with my microarray experiments and hope
that I am not the first one to see this.
Aminoallyl-dUTP-containing PCR products are used for hybridization
with oligo microarrays after in vitro labeling with Cy3/Cy5; the
slides are scanned using GSI Lumonics ScanArray 4000. When I switch
the dyes there is a significant difference in results (e.g. complete
reversal for many spots).
My guess is this is the result of leakage of Cy5 signal into Cy3
channels, and I hope there is a way to adjust the detector to cut
that. Alternatively, there is a significant bleaching of the second
dye while the first one is scanned (I did not chenge the order of
scanning). However, these are just guesses, and any ideas and
suggestions will be most appreciated. Please use my email
(levenson at northwestern.edu).
If there is enough interest I can summarize the responses.
levenson at northwestern.edu