1 reply to this topic

[jvollme]

Hi, I'm a little confused about how the pfaffl-method for relative real-time quantification is commonly used.

Most people state how important it is to have nearly identical primer efficiencies, and that they should lie between 95% and 105%. Often the pfaffl-calculation-method is cited:
(E_target)^{deltaCT target
   ratio=    ---------------------------------}
  (E_ref)^{deltaCT reference}
however it is usually abreviated to the deltadelta-CT-Method (2^{-deltadeltaCT}) under the assumption that the primer efficiencies are identical and close to 100%.

My question now is: Isn't the whole point of the pfaffl-Method NOT to assume identical primer efficiencies?
Shouldn't you be able to calculate relative abundancies accurately with this method, even if the primer efficiencies differ strongly, and even if they are far from 100%?

I want to compare the expression-rates of 4 different genes, however I calculated 4 different and low efficiencies for them (92%, 83%, 87% and 89%). However i am under strong time pressure and do not have the time and resources to optimize the assay further.
Can't i just calculate the abundancies in these qPCR-reactions using the original Pfaffl-method (correcting for the low and differing efficiencies)?
Is there any reason why the efficiencies should be over 95% and almost identical even though the pfaffl-method corrects for such differences?

[Trof]

Yes, the whole point of Pffafl method is not to assume identical (or 100%) primer efficiencies. But having an efficiency outside the common limits 110% - 90% means the reaction is not working well and such assays are not recommended, not only for having different efficiency, but because something is wrong with it.
Pffafl says it's important to account for all slight changes in efficiency, the delta-delta people say that you rather should design all your primers to be very close to 100% and then don't care. None of them says to use primers with bad efficiency, but if you do you should rather calculate with Pffafl.