Poor data from Cy3 channel

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Poor data from Cy3 channel

Postby LadyGiardia » Oct 17 2008 4:07 am

Hi there,
Can anyone help? I'm using TIGR arrays for CGH and the data from the Cy3 cannel is awful. I have managed to slightly minimise the high background but this is still a problem.
The Cy5 channel is excellent; clear good spots, low background, totally different to the Cy3. Others in the lab using the same dye do not have a problem with it. I've checked dye incorporation: fine; DNA concentration: fine; Different washing buffers with and without DTT, Dye swaps: test and control DNA fine on Cy5. Everything I do is the same condition as with the Cy5 labelled DNA but the data from each channel is so different. How can I work with this data? I've done a control ref:ref slide so hopefully I can normalise the data on both channels for the control and apply that to the test:ref slides, but this is such a pain. It should work, but from the scan images it looks like the cy3 labelled DNA has had trouble binding.
I am totally vexed!
Does anyone else have a problem with the Cy3 channel?

Thanks
LadyGiardia :(
LadyGiardia
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Postby dalle » Jan 30 2009 4:50 am

Hi,

did you find a solution for this problem, because I have the same problem. I´m using epoxy slides from corning.

Thanks for helping

dalle
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High background in Cy3 channel

Postby LadyGiardia » Jan 30 2009 6:28 am

No unfortunately not, even after correspondance with the supplier the amino silane coated slides just didn't cut it and background was so high it masked the signal. But the previous slides I had did have an epoxy coating and I found no problem with them when I used 50% formamide in the hybridisation buffer, and omitted the DTT. Also make sure that your washes pre-hyb are vigorous and don't let the slides come out of the liquid as they dry very quickly. Same goes for post-hyb washes actually.
Type again if you need anymore info. and let us know how you get on!

best wishes
LG
LadyGiardia
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