Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum
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Hello, some diseases induce some enzymes, which are inactive during healthy period of life. How would be deltadeltaCq calculation in case that presence of control results is impossible.
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The non-disease state will be a good control. Although the level of enzyme in non-disease state may be very low, it can still give you a deltadeltaCq value (40 cycles of qPCR can amplify a single molecule). A good example is heme oxygenase 1, which is an enzyme that is induced by oxidative stress. Fold induction can be huge.
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How meaningful is that huge induction? When PCRing beyond 30 cycles, often the sample replicates start having more divergent Cp's. Using these numbers as the denominators to divide the positive samples by the effect of these variations are amplified. Even when the quantification is accurate, there might not be any biological difference between 1 and 20 molecules of an mRNA in a cell, but when this Cp becomes the denominator you then see a twenty fold difference in an already huge induction. It seems to make sense to me to always show the "uninduced" or "non-disease" number along with the induction, so that there's some indication of it's significance, and maybe an asterisk to indicate which samples have an induction that can't accurately be calculated because the "uninduced" level is too low. I'm not sure the official way to handle this.
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