I'm using a CFX96 and when the machine automatically picks a Ct, I get very bad efficiency values (~250%).
However, when I manually move the threshold for Ct much lower, I can get:
r-squared = >0.99
efficiency = ~105%
There is no signal from the negative control or NRT.
Am I allowed to do this? Can I just manually adjust the threshold until I get good values? To get good efficiency, I do have to set the Ct very low on the amplification curve.
Hello
Yes you are allowed to do so and manually adjust the threshold line.
By definition the cycle threshold of a reaction is defined as the cycle number when the fluorescence of a PCR product can be detected above the background signal.
As long as you stick to this definition you are allowed to lower your ct threshold.
Sometimes when running many samples in a one run the device aromatically detection cannot compute ct very well and you have to change it manually.
It's difficult to judge without seeing the real data. In principle, you can shift the threshold if the curve shapes allow this over a wider range without major changes in the relative Ct differences and efficiency. For this to be allowed, the curves should more or less be parallel and the changes would be not so big compared to the automatically calculated values.
If you have very different slopes in your curves for different samples that could indicate a problem with the PCR efficiency (inhibition?) in those reactions. Then it may not be OK to just shift the threshold to a value that is by chance in the range that you want it to be.
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