reaction efficiency - (Apr/10/2011 )

Hi,

I'm using the comparative quantitation software of Corbett do you now which is the range for the efficiency reaction? I read that a value > 1.6 is ok..why?It is not too low?
Another question: could I compare reactions with different efficiency? For example an efficiency (for the target gene) of 1.75 with an efficiency of 1.86 (for the reference gene)? I read that with the comparative quantitation software I can compare different reactions because it calculates the efficiency on the basis of fluorescence increase in the PCR exponential phase, it is right?what does it mean?It means that I can compare reactions with different efficiency?
And which is tje advantage to calculate the concentration using reaction efficiency?
Thank you very much!

First to say is I don't have any experience with Corbett, so I don't know what its software does and does not.

Basic delta-delta Ct quantitation method (and the underlying basic qPCR theory) calculates with assumption, that PCR reaction(s) have the same 100% efficiency, in this case equal 2 (which means product completely doubles in one cycle). These ideal conditions are almost never met, because it is a real life and not a theory. So a new method by Pfaffl was proposed how to normalise gene expression between two genes that have different efficiency.
So yes, not only you can use efficiency when it's different between genes, but more, you should do it, bacouse if your reactions differ in efficiency from the ideal model, your results won't be accurate.

I'm not sure about what comparative quantitation software are you talking about, but efficiency is usually calculated from dilution series. There is a method how to calculate the efficiency from the shape of the amplification curve, as PCR Miner does, but these are slightly different from those calculated with dilution series, but it's not sure which one is better. Also dilution series can be affected by potential inhibition of more concentrated template and that's the case when the efficencies go higher than 2.

And if I remember it right, range of suitable efficiencies is +/- 10% from 2 (or it was 5?), out of this range it is generally considered not a good working reaction.

Thank you very much for your help!and what about the efficiency calculation that is = 10^(-1/slope) do you know how this equation is obtained? thank you!

Another question, I read that with the mathematical model of Pfaffl, the efficiency is the same with different concentrations of input DNA; but if I use a lot of DNA the Ct and the exponential phase begins early, on the other hand if I use low concentration of DNA exponential phase begins late...so why usually it is preferred that the exponential phase begins around 15-20 cycle?why It is not good if the exponential phase begins too early or too late if the efficiency doesn't change?
Thank you very much!

genetics3 on Mon Apr 11 08:54:17 2011 said:

It's from the regression stright-line of dilutions, which is defined as: y = kx + c
where:
x = log10 of concentration
y = Ct
k = slope
c = intercept
but I can't tell you more, I'm not exactly proficient in the mathematics of qPCR.

genetics3 on Mon Apr 11 09:52:09 2011 said:

If you have Cts to high, there is small numbers problem coming in and stochastic events happening, that will cause a bigger variation between replicates. I'm not sure why exactly the other extreme is also bad, but it could mean excess template, which may affect the reaction, I don't think when they say the efficiency is same in different concentrations, that they mean extreme values. But I met a more broad range from 15 to 30, that is considered quantitable.