Variation in Standard Curves - request for an explanation.

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Variation in Standard Curves - request for an explanation.

Postby MBDM » Jul 26 2018 9:36 am

Hi all, maybe I have the IQ equivalent to an inferior form of pond life, but could somebody explain this diagram to me. Are both slopes being generated using the same threshold value or is the threshold value different for each curve?, i.e. has the 100% efficiency curve a different threshold value setting to the 78% curve? Intuitively, I cannot understand how the efficiency for an assay that is less than 100% can sometimes have Ct values greater than (which i would expect) and sometimes less than (hard to comprehend) an equivalent assay having 100% efficiency and the same amounts of starting material. :?

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Re: Variation in Standard Curves - request for an explanatio

Postby 29yrsExperience » Jul 30 2018 5:50 pm

First, the Figure does not mention threshold, so it is most likely that it is meant to depict a single experiment (with two targets, thus two standard curves) in which the threshold was the same for the whole thing. Reading the text accompanying the figure ought to clarify this. Threshold might be irrelevant here.

The threshold is calculated based on the mean background fluorescence (or baseline) in the early cycles (usually 3-15) before any target in the experiment starts to amplify to detectable levels. While the threshold will affect Ct values, Ct values are not used to determine the threshold. However there are times when you may need to adjust your threshold, and this can indeed result in a different slope for your standard curve. There is a decent explanation here ... holds-.pdf

As for wrapping your head around the apparent contradiction shown in the figure: it seems counter-intuitive to me, too. But the graph itself demonstrates how it could be true in certain circumstances.
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Re: Variation in Standard Curves - request for an explanatio

Postby r.rosati » Aug 02 2018 4:33 pm

The source material for this figure mentions,
"In Figure 5, two samples (X and Y) amplified under low and high efficiency conditions show different Ct values for the same target concentration. In this example, although the high-efficiency condition (the blue curve in Figure 5) gives a later Ct at high concentrations, it results in better sensitivity at low target concentrations."
I agree with you that the figure is a bit unusual, in that both high-copy and low-copy data points are affected by the alleged low efficiency of the reaction, and there's a "magic copy number" that is unaffected in the middle of the curve. Although I can't rule out such a thing,I don't have at hand a similar situation in practice.
For example, if the low efficiency was due to PCR inhibitors, then the Cq of the high-copy tubes would be higher than expected; but then the Cq of the low-copy tubes should remain similar to a 100% efficiency reaction, since the inhibitor has been diluted.
Similarly, an error in the dilution of standards would affect the low-copy data points (possibly resulting in higher than 100% efficiency), but it shouldn't affect the first data point.
Perhaps other factors might create this situation... Wrong annealing temperature perhaps? But I've never seen it happen.
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