Microarray problem with switching dyes
jongh711 at planet.nl
Wed Mar 9 20:41:57 EST 2005
there are a lot of reasons why the Cy5 signal can differ from the Cy3 signal.
They have different quantum eff. for a start. One of your reactive dyes maybe
off. There maybe too much ozone in the air which affects Cy5 more than Cy3. I
think that there is very little chance that it has something to do with your
scanner. The Axon scanner we use comes with a piece of fluorescent material to
calibrate the two channels , maybe you can obtain that from Axon . You can buy
Cy labelled random nonamers to check quickly both the scanner and the
microarray.There will always be a difference in the two channels for the mean of
the fluorescence which is usually taken care off by propper normalisation.
Please look here for a more dedicated forum :
"Victor" <levenson at northwestern.edu> wrote in message
news:52c09469.0503091140.3fe7a847 at posting.google.com...
> Dear all,
> 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.
> Thanks much,
> levenson at northwestern.edu
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