HEX vs. Cy-5 - (Sep/25/2009 )
Hi All,
I have a taqman assay that uses a HEX probe. In multiplex with a FAM probe, I have been having a hard time getting consistent standard curves with the HEX probe. The curves with the FAM probe are fine. I was thinking of changing the HEX probe to a Cy-5 probe.
Could this help clean up my multiplex assay? Any reason why I might not want to do this?
Thanks
Hi RatRace2000,
I am not surprised that you are having problems with a HEX assay. Unlike FAM assays the amount of energy you get from a HEX assay is significantly lower so the assay needs to work a lot better to get the same quality of signal as a FAM assay.
Switching to a Cy-5 assay may work, but of course it all depends. You may be better off re-designing your HEX assay, it all depends on how good the assay is (is it really an issue of the HEX dye or the assay itself?).
I assume you realize that which dyes you can use depends on the instrument you have. If your instrument has not been calibrated for Cy-5 then there is a good chance you will not get much better data with it. It may still work, but not that well.
Good luck
What is the exact problem? You may find that like the previous poster said, the problem may lie with the assay, not the fluorophore. For example, the HEX assay may be suffering from competitive inhibition from the FAM assay, whereby the FAM assay outcompetes the HEX assay for the reaction reagents. You could easily test this by running the two assays parallel in singleplex and in duplex with the same standards, as see if that makes a difference in Ct value/quality of curves.
It could also simply be that the HEX assay primers negatively interact with each other, or with the oligos of the FAM assay.
FAM is a strong emitter, so quite commonly it will bleed through to other adjecent channels. You may want to try some colour compensation to filter out such bleed through.
We have also found that the Cy5 tends to be a weaker emitter than HEX.
RatRace2000 on Sep 26 2009, 01:55 AM said:
I have a taqman assay that uses a HEX probe. In multiplex with a FAM probe, I have been having a hard time getting consistent standard curves with the HEX probe. The curves with the FAM probe are fine. I was thinking of changing the HEX probe to a Cy-5 probe.
Could this help clean up my multiplex assay? Any reason why I might not want to do this?
Thanks
Unagi on Sep 28 2009, 02:18 AM said:
I inheritted this work from someone else and it looks like your suggestion of running the assay in simplex is where I will start.
When we run the assay, we find that the HEX channel (on a std curve) is seldom repeatable and often not very efficient <80% unlike the FAM assay also present.
Thanks, the info was very helpful.