Shanghai Jiao Tong University
Question
Asked 9th Feb, 2021
QPCR troubleshooting - what could cause an increasing ROX signal?
Hi everyone,
I'm looking for some help troubleshooting a recent qPCR issue I've encountered. I am using fluorescent Taqman probes and a qPCR master mix that includes ROX as a passive reference. My understanding is that the signal from ROX should be relatively flat when looking at a plot of fluorescence vs cycle number (assuming that there are no pipetting inconsistencies, evaporation, bubbles, etc. in particular wells). I am seeing a fairly significant, gradual increase in the ROX signal in every well. In fact, some of our reporter/target dyes are also showing a similar increase in the initiation/ground phase of the curve.
Can anyone offer some insight or advice on this issue? I would appreciate any help! Thank you in advance.
Most recent answer
Any idea whether ROX is compatible with taqman probes for multiplexing through Stepone or LC96?
All Answers (4)
Justus-Liebig-Universität Gießen
Possibly some form of eximer production. It may also be the result of changing buffer conditions (pH). I don't see a particular need for "trouble shooting". In fact, the passive reference is perfectly demonstrating the amplification-independent signal drift if should correct.
Institute of Agrophysics, Polish Academy of Science, Lublin, Poland
Do you try at different concentration of probe? If you run the two probe separately, not together, do you see the same?
Ferdowsi University Of Mashhad
Hello
What is your qPCR instrument? Not every instrument need a passive reference dye! For those needed ROX as a reference, you must set the program to read the ROX as the reference, so it wont show up in your actual reading.
Good luck
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