We are having trouble to determine the threshold line of qPCR in a ABI 7500. We always used automatic settings for determination of the threshold line, but recently the software is setting the threshold line below reaction background. Do you have any idea about what is causing this problem and how we can fix it? Attached is following an amplification plot of the wrong threshold. Thank you.
Do you have any trouble with your non Template control? Have you changed your ROX reference calibration recently ?
Perhaps it’s a good idea to recalibrate all the used prob…
Do you have any trouble with your non Template control? Have you changed your ROX reference calibration recently ?
Perhaps it’s a good idea to recalibrate all the used prob…
Non template control presented amplification at late Cts (Ct>36). Another experiment using SYBR Green presented normal threshold, but FAM probe presented the wrong threshold.
Actually, we are planning to recalibrate the system.
If you still need the data from such a run, 0.1 manual dRn threshold could be used to apprehend reasonable Cq values here. If your FAM is being counter-balanced by TAMRA on the 3' end - you may have higher initial unquenched signal (TAMRA doesn't totally FRET-quench FAM). MGB-NFQ on the 3' end is much quieter. BHQ is often better as well. If this is not a calibration issue, it seems like you should not be afraid to use manual threshold settings (memorize them for each different target from same-tissue-same-isolation-method-same-master-mix protocols/work-flows). In your image, the machine is being fooled by the orange line intersecting the thresh at ~7 cycles. The thresh line needs to be gently nudged out of its confusion by the user here ... to about 0.1 dRn.
I agree With Jack reply, manual threshold could be used.
But I add to add it's not regular to have amplification in your Non Template Control, especially if you use Taqman technics…
I have the same respons as Jack,
A lot of the time (mostly) I set the treshold manually on about 0.1 for all my targets,
Why? If you are running multiple plates and doing delta Ct analyses and your machine sets your threshold for gene A in the first plate on 0.05 and gene B on 0.07, but on the second plate,(since you might have used a different mastermix, even pipetting differences, a whole lot of things might change your reaction effeciency a bit). Your machine might put the threshold for gene A on 0.07 and the threshold for gene B on 0.07 again. Your delta delta Ct for both plates will be different.
If you put the threshold manuallly at 0.1 (works for about 99% of the assays). Your delta delta Ct will be very nice and stable from plate to plate
Hi Tomas, I suggest to use the "LinRegPCR" software to analyse yours qPCR raw data. You have to set the Relative Lights Units (RLU) without baseline substraction and then export the file to Excel. Hence, you can run the LinRegPCR software, which will read your Excel file.
Hello Tomas,
It's fairly rare occurrence that our software will have that issue, and indeed it might be due to your FAM assays exhibiting a higher than normal level of background fluorescence, leading to the algorithm getting 'confused'. Recalibrating the instrument might help, but you can definitely designate the threshold manually for this run, and your data should be fine. If you'd like someone from our tech support team to look at your file, feel free to send it to: [email protected].
Have a good day,
Kevin
I would like to thank you all for your answers and for trying to helping solving our problem.
We recalibrate our system using a new Spectral Calibration Kit as recommended by Laurent and other researchers, and we performed a new qPCR and the same problem occurred. After that, we contacted Life Techlonogies support and I explained our problem. We discovered that the real problem actually was in the FAM probe. Life Tech is actually providing a new probe for us.
We think that a new probe, our problem will be solved. Thank you Kevin for your answer and help.
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