Calculating efficiency of raw data - (Sep/03/2006 )
How does one calculate the efficient of a PCR reaction? I am trying to follow in the footsteps of Peirson et all (2003; doi: 10.1093/nar/gng073) but fail miserably with the efficiency calculation.
This is what I do for each well:
Find the maximum fluorescence value of the well
Estimate the background noise by taking the standard deviation in fluorescence of the first 10 cycles
Find the midpoint by
midpoint = noise * sqrt(max/noise)
(eq 2 in the paper)
Find the lower and upper limit of the linear regression by
lower = midpoint/5
upper = midpoint * 2
Then I make a lineray regression using 10 base logarithmic values of the fluorescence
Using the intercept with the y axis and the slope of the linear fit of log10(y) vs. x I find the Ct value:
ct = (log10(threshold) - intercept)/slope
So far so good :-D
The error Is estimated by eq 4 in the paper:
efficiency = 10^(1/slope) - 1
And should be between 0 and 1. However, I get values like 38.... and thus a not so correct estimate of the starting fluorescence
Plaffl uses 10^(-1/slope) for the efficiency estimate, which takes a value between 1 and 2
The slope of what is used in the efficiency estimates?
Is it a mistake that the minus is missing in the Peirson efficiency estimate?
Should the slope of the linear fit of log10(y) vs. x be transformed?
Why do I want to do this?
Not all of our standards work all the time, and at times one would like to compare without standards (I know the discussion of relative vs standards). My plan is to estimate the starting fluorescence my self, and based on the available data, do various calculations across all datasets, and who knows... maybe one day there will be a package for R :-D
Thanks in advance for any help and suggestions
You are not being ignored. I think that most people here are like me and have software on our real time machines that automatically calculates the Ct values, so we don't have to do the first part of your method.
What do your Ct values look like? Are they believable (ie something like somewhere between 5-35?) If not, then I'd say the problem is in the first part of your method.
The method describe (above) appears to be intended for measuring the efficiency of a PCR reacton in a single well (calculating baseline noise and slope of curve). The problem with any method like this is that PCR efficiency has often declined dramatically once the reaction is fluorescent enough to be visible to the instrument.
A much better way to calculate PCR reaction efficiency is to go from the slope of the standard curve (Ct value versus log concentration). This method is much more robust, here's an example of how it is done (formula at bottom of page):