Real time 70% efficiency and DDCt - (Jun/11/2009 )
Hi Everyone
I have a question for you. I'm doing a real time and I'm testing two different genes. The reference I'm using is actin. I want to use the 2^-DDCt method (Livak and Schmittgen) but I don't know if I can use it. I did a serial dilution of my cDNA and I plotted the log cDNA dilution versus Dct (for both genes with the reference) fot testing if two amplicons have the same efficiency. The slope is <|0.1| so they have the same efficiency. Efficiencies were then determined from standard curves generated by serial dilutions of the cDNA sample and estimation of the cycle threshold at each dilution. Efficiency are 1.67 for both sample genes and 1.71 for the reference (R>0.98).
So the assumption that efficiencies must be approximately equal is confirmed but I don't understand if I can use the formula 2^-DDCt or if I have to use same correction factor.
Thank you
Hi cece,
I think 1.67 (sample) and 1.71 (reference) is not good enough to be called equal. For DDCt method, the efficiency should be same...
Nonetheless, you will get a similar trend with this but not the exact reality. R=0.98 is not good enough too in realtime pcr....
i suggest use both ddct and standard curve method and compare...
let me know as well:)
cece23 on Jun 11 2009, 10:52 AM said:
I have a question for you. I'm doing a real time and I'm testing two different genes. The reference I'm using is actin. I want to use the 2^-DDCt method (Livak and Schmittgen) but I don't know if I can use it. I did a serial dilution of my cDNA and I plotted the log cDNA dilution versus Dct (for both genes with the reference) fot testing if two amplicons have the same efficiency. The slope is <|0.1| so they have the same efficiency. Efficiencies were then determined from standard curves generated by serial dilutions of the cDNA sample and estimation of the cycle threshold at each dilution. Efficiency are 1.67 for both sample genes and 1.71 for the reference (R>0.98).
So the assumption that efficiencies must be approximately equal is confirmed but I don't understand if I can use the formula 2^-DDCt or if I have to use same correction factor.
Thank you