Hi all
I am reading QIAGEN's article (https://www.qiagen.com/es/resources/faq?id=2d04d81f-ef1f-4d7e-aaa5-2f30767445b1&lang=en) about the delta-delta CT method of quantifying qPCR. It says that it would not require standard curves but then you have to perform a validation experiment to test the amplification efficiency but isnt that the same as a standard curve?
The "problem" with delta delta Ct is that it assumes 100% efficiency. Something like the Pfaffl method allows you to set your amplification efficiency if you know it. You don't have to run a standard curve for ddCt, but it is helpful so that you know for sure whether or not your efficiency is even close to 100%.
In order to determine amp efficiency, you will have to run a standard curve and determine your slope. There are calculators online to help determine the efficiency once you have the slope available.
Hi Amanda,
Thats where I am confused about, many qPCR guides talk about ddCT method not needing a standard curve but when it comes to measuring the amplification efficiency of the ddCT experiment, you still need to run a standard curve anyway. It's so very confusing.
Is there any way to get around the amplification efficiency? I am using Taqman probes
I'll be honest, I've never worked in a lab that actually ran an amp curve for ddCt. If you're using TaqMan, just watch your amp curves to make sure they look nice, make sure you're running all of your controls, and run everything in triplicate. I even had an entire semester-long class on qPCR and we never ran curves for ddCt. Especially with TaqMan, if you're running everything appropriately your amp efficiency should be high enough.
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