Hi, I want to analyze my qPCR (gene expression, mechanical tested and not testet) results, and have to normalize them to my reference gene GAPDH. I don't have a standard curve.
I have looked at two methods, and can't decide if I should use one of them, or both.
1) is the 2^(delta delta Ct) which gives a fold between the tested or not, and normalized to GAPDH
2) is just dividing the sample Ct with GAPDH Ct and then compare the two results from tested and not testet samples.
What do you think?
Method #1 (2^(delta delta Ct)) is the standart analysis to get fold changes between your conditions.
In general, a standard curve is needed to evaluate the primer efficiency. If it has been previously investigated, then in most system, you are allowed to enter the amplification efficiency and calculate the fold-change using the Delta-delta Ct method (1st method). You can refer to the Biorad Real-time PCR handbook which clearly indicate the calculation for relative expression
As Martin Chenal has pointed out that the Livak and Schmittgen method (2–∆∆Ct method) is the standard way for gene expression profiling. You would need to normalize the values vis-a-vis your reference gene first and then with respect to the calibrator (Control sample) to get meaningful results of your relative gene expression analysis.
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