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I always add the Taq to the mastermix. First it makes the handling easier and it avoids pipetting steps which can cause contamination and can also be forgot. Then the enzyme is very stable and will even tolerate room temperature without problem. Since we are going to heat this 30-40 up to 95°C, so this is clearly not problematic. Since the reaction mix is ...


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First step is the calculation of efficiency, denoted by lets say $E_{gene}$. See this post for calculation of primer efficiency. So the fold change for that gene will be calculated by $E_{gene}^{-\Delta Ct_{gene}}$ Where: $\Delta Ct = Ct^{treated} -Ct^{control}$ But these Ct values are not normalized. For normalization, you take some reference gene ...


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This topic could probably have it's own StackExchange, there is so much info and trial and error done with rtPCR. Here's my 2 cents: Your adjusted calculation does not normalize your data, it just subtracts the 'background' or control. For expression studies this is not good enough. Normalizing data for gene expression must involve setting it to a scale of ...


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As I mentioned in your previous post. If your reference gene undergoes changes then replace it with another reference and if even that doesn't work out then use a spike in. You must normalize. Interpreting the raw values can be erroneous- there could be a loading bias. Don't think much- repeating RNA isolation and/or qPCR is not a very hard job. And as Chris ...



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