delta delta Ct method - Real time PCR (Jun/26/2005 )
2^(-delta delta Ct): for using this formula then we have to get the same PCR effiencies for both the target and reference genes. Do the PCR effiencies have to be 2 or can it be at other PCR effiencies too? And if it can be used at other PCR effiencies then how reliable is this formula since it is based on 2 as the PCR effiencies?
I am sorry I don't quite understand your question since I don't know which formule you mean.
But overall it is handy to know that the formule of calculating the PCR efficiency is based on the concept: after each cycle the number of amplicons is dubbled. A binary relationship between the copy number and the cycle.
Since our daily calculation are based on decimal, and 2^3.32 is 10. The '3.32' is most popular used to indicate the PCR eff. Which means after 3.32 rounds the number of the amplicons should be 10 times more if the efficiency is 100%.
Knowledge makes life better
try this article:
Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-8.
it explains the equation and when to use it. i found it helped me alot.
Theoritical PCR efficiency (I think amplification efficiency is a better word) is 2. But, in most reaction, the efficiency is less than 2. That is why we should take Ct value from linear rage of the graph of dRn vs. cycle number. But we assume that amplification efficiencies of samples on the same 96well plate are same. So based on this premise, you can use ddCt method for any sample on the same plate. But for other reaction, we can not assume that the reaction has the same amplification efficiency as that of previous reaction. So we need to include reference sample in every PCR reaction even we use same reference in all the reaction
PCR efficiency is calculated : Eff=(10^(1/slope))-1. a slope of 3.32 significates an efficiency of 100% (DNA quantity doubled each cycle), if you find 90% that means that 0.9 DNA molecule is generated each cycle from 1 DMA molecule . I don't know how you can find an efficiency of 2! It is a non sense, an efficiency is always a percent!
Efficiency (100%, 90%...) does not matter for deltadeltaCt method, the important thing is that efficiencies of the reference gene and the target gene should be very closed . For controling this, you generate a graph : x axis : log of amount of cdna, y axis : Ct(target gene)-Ct(control gene). The slope of the tendancy curve must be <0.1. If it is, you can perform deltadeltaCt method else you can't.