PCR efficiency calculated by Linreg - (May/18/2010 )
I use Linreg to convert my data of RT-PCR. The program calculates a PCR efficiency for each well and a average PCR effciency
for the whole run.
Now, I have new primers and I run dilutionseries to calculate the PCR efficiency.
I've trying to design new primers for a number of months now and finally have primers that work...but
the Linreg program gives a good PCR efficiency (around 97%) but when I calculate it myself I only have around 60%.
Normally the data from the program is close to my calculations so Ive checked my calculations a number of times but
I can't see the fault...
The melting curve was good, the negative controls were negative...
So I finally have primers that work, but good enough??
Can somebody help?
What do you mean with "your calculation"? Is it standard curve slope?
I plot the dilution transformed to a log to the Ct values of the dilutions. Then I calculate the slope of the line and transform it to a percentage...
Yeah, that's the standard method to calculate PCR efficiency. You should believe this method more than curve-fitting methods. You probably did your dilution series wrong.
Vortex vigorously when diluting samples.
Use DNA carrier such as glycerol if your concentrations are low.
Ensure you are measuring Ct's for standard curve between 10 - 30 cycles. Discard results after 30c.
Have your standard curve span at least 5 logs of concentration - i.e. 10e1 to 10e6.
Run in triplicates.
I was also using various curve-fitting models and it was giving me funny values too. Although I like that approach I think it is not robust enough to give you reliable estimation of PCR efficiency. Maybe one day after it will be standardized by some large qPCR cycler manufacturer it will be better.