Are you stuck trying to distinguish real signal from background noise in your qPCR data?
A common question about qPCR data is how to distinguish true signal from background noise. Today, we’re going to provide some useful tips on how to tell whether you are looking at real signal or background.
Sometimes when you are running qPCR experiments, it can be difficult to distinguish real data from background signal, leaving you to wonder whether your data is real.
Here are some parameters to check to determine whether you are looking at background or noise:
First, make sure to run No Template controls to compare to your experimental samples. If your sample signal looks similar to your No Template Controls, it’s most likely background.
Also, check your replicates. If the Ct value varies among the replicates (ex: 36,37,40), then it is likely not real signal.
You can also do a reality check on your Ct values. In general, a single copy detection occurs by a Ct of 37 in a 20 uL qPCR reaction. Any signal seen later than this is not likely to be real amplification.
Often, you can easily tell whether you are looking at true signal or noise by looking at your data in the instrument or cloud software. Here’s an example using the Thermo Fisher Cloud Design and Analysis Application. This well is questionable. If we change our plot to the linear view, we can see that this curve is actually flat, and no amplification is occurring. We can see this in the multicomponent view as well. This view shows the curves of each dye in the reaction at each cycle, and is very useful for troubleshooting. In this way, we can check the curves for exponential amplification, which indicates real signal, as opposed to a linear upward drift which can be probe degradation at the end of a run.
We can even overlay the wells with our NTC samples, and see that there is not much difference. So we can be confident that those were wells do not represent real signal.
Finally, you can also make sure the threshold is going through the exponential phase of the curve. For example, here you see that we have Ct values at 28 and 36, but the curve with the Ct value at 36 is from the plateau, not exponential phase of the curve, so this is not likely real signal.
So, the next time you look at your qPCR data, try some of these useful tips to determine whether you are looking at true signal or background noise.
If you have more questions on qPCR data analysis or any other qPCR questions, remember to Ask TaqMan and submit your questions on our website www.thermofisher.com/ask
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