absolute Quantification despite different efficiencies - (Oct/31/2005 )
I'm going to absolute quantitate seriously degraded DNA in the LightCycler. The DNA shows persistent low amplification efficiency, while the cloned plasmid standards behave optimal. It wasn't possible for me to purify it more.
My question is now, if it is possible to work with these DNA-samples. Is it essential, that sample and standard have the same efficiency? Or is it possible to calculate the efficiency based error to get the correct DNA-copy content in the sample. And, are there any publications that deal with this topic?
Thanks for your help
It depends what you want to do with your DNA and how badly degraded it is. For real-time PCR, smaller amplicons are generated so even degraded DNA can be analyzed okay.
The DNA runs in the Lightcycler. But serial dilutions of it showed low efficiencies, probably due to present inhibitors, that I wasn't able to remove in the purification. There are various publications, that deal with different efficiencys at relative quantitation. Is it possible to get the exact DNA amount with absolute quantitation, despite different efficiencies between sample and standard? By comparing the efficiency of standard and sample, you can calculate the error (i.e. 170% at cp). Is this trick allowed or do I have to find a way to increase the efficiency rate of my samples?
Thanks for help
I'm still looking for an answer to my problem. Can anybody say if this calculation is allowed? For example, if my plasmid standards have an amplification efficiency of 1.9 . But my quantified sample has an efficiency of 1.85 at, e.g. a cp of 23. Is it possible to get the correction factor by comparing the fictive copy number, the sample would have at the optimal efficiency (1.9) with the measured copy number?