Hi,
I have a question on RT qPCR:
until now I used Livak method to analyze my qPCR data. I normally use 8 different reference genes and determine the three most stable ones. Then I use the geometric mean of Cq of these three reference genes for 2^-ddCq method.
Now I want to include PCR efficiency values via Pfaffl method. I am able to do the calculations referring to each single reference gene. But I don´t know how to combine more reference genes in the analysis.
The formula contains the efficiencies for target and reference genes. What is the efficiency for the control gene if I use 3 different ones?
- hi Verena
- If your efficiencies are within 5% then the value you plug into Pfaffl analysis is also the geometric mean
- Very good question by the way !
I recomend you this paper : Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8(2):R19. doi: 10.1186/gb-2007-8-2-r19. PMID: 17291332; PMCID: PMC1852402.
Here, the authors use a modification of Pfaffl Method to use more than one reference gene.
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