Microarray problem with switching dyes

[Jose de las Heras]

"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).

you mean that a bright cy3 spot in one direction, then turns out to be 
bright cy5 on the dye swap?

unusual, and I don't think it has anything to do with the labelling, but 
perhaps your samples. Do you see the same thing if you take one sample, 
split it in two, and label both with different dyes?

> 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

I really doubt you're seeing leakage, as cy3 and cy5 have negligible overlap 
in their emission spectrum, in the way they're used.

I can't imagine why you'd see the reversal you talk about. I'd blame the 
samples. You could try to label a single sample, divided in two, with both 
dyes and hyb. You should get the same(ish) results on both channels. It's 
useful to try a few tests like that where you know what to expect, before 
you embark on your real experiments. If you can spike your samples with 
something you know it's present in a few spots, even better (in combination 
with the above).
Do you have access to another scanner? You could try to get your slides 
re-scanned elsewhere and see what they look like. Although most protocols 
recommend scanning immediately after hybridisation, if you keep your slides 
in the dark and dry, they stay good for quite some time. I just gave 
somebody a slide of mine that was first scanned in mid-december, and my 
slide was still very bright (better than their own, in fact :-) If you scan 
it somewhere else and you get the same effect you can rule out any problems 
with your scanner.

Good luck!

Jose