Hi ResearchGate community,
I have been trying to learn more about the optical differences between block-based real-time PCR machines like ABI StepOne versus rotor-based machines such as MIC or RotorGene systems.
I understand that some systems rely on ROX as a passive reference dye while others state that it is optional to incorporate it and others do not need such a factor at all.
My question is if you add this fluorescent dye to your master mix, would it interfere with the detection when it is being amplified using one of the systems that do not need such normalization?
Highly appreciate any insight in this regard.
Best,
Negar
Hi,
I observed that too much ROX (for example in adding ROX in a mix that already contain a sufficient amount of ROX... >_
Thomas Guiraud Hi Thomas,
Many thanks for sharing your experience.
I wonder if you've got a ROX probe in one of the channels, would you still need to add ROX dye as the passive reference agent for instruments such as ABI stepOne that require high ROX normalization?
If yes, would it be ok to have a universal master mix with ROX (maybe at a medium concentration) for all real-time machines regardless of their ROX requirement?
Best,
N
I don't think it's a good idea to use ROX for both a probe and the passive reference : you would loose the benefit of the ROX normalization. If you don't want to do this normalization, I think the ROX present in the mix can only increase the background and consecutively, the baseline and the Ct. The more ROX you add, the higher would be the background.
Concerning the ROX content in the PCR mix, according to the teaching of my unfortunate experience, I wouldn't be "creative" and would strictly follow my machine requirement (it wasn't a StepOne but an Agilent MX3000P).
Many thanks Thomas, Thomas Guiraud
I already have ROX in my PCR master mix as my assay relies on probes labeled with FAM, HEX, Cy5 & ROX.
What I am not sure about is would it be fine to just ignore the fact that some machines such as Agilent (which is the one I have been experimenting a bit with) or StepOnes require ROX for normalization and uncheck that option in the setting or it is necessary to include another dye for that purpose other than ROX.
On the MX3000P machine from Agilent, you can make two choices : you can uncheck the ROX signal registration (and you won't retrieve any ROX signal) or you can register the ROX signal and select a baseline determination strategy without a reference dye signal. I suggest you to register the ROX signal (because you're using a ROX probe) and to not choose a baseline determination strategy using a reference dye. Even if you don't use your ROX probe, to my mind, it's always a good strategy to record all the signals you can and make the choice thereafter to not use these data. The MxPro software is rather convenient for that.
I can't tell anything concerning the StepOne machine and software.
Thomas Guiraud Fabulous Thomas,
Many thanks for your kind support.
Guess I am beginning to get a more clear view of it all as I keep experimenting.
Best wishes for all you do,
Negar
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