Hello everybody,
I'm performing absolute standard curves (with a known concentration) in a new qPCR machine. Unfortunately, I have a problem reaching efficiency above 85%, although I had high efficiency with the same primers in our old qPCR machine.
While consulting with the machine company expert, I was told that in an absolute standard curve, efficiency doesn't matter; it only matters in a relative standard curve.
Have any of you know something about it? I can't find any paper that approves it, but I see that the efficiency refers mainly to the relative standard curve.
Thank you for your help
Efficiency is an important parameter in quantitative PCR (qPCR), as it affects the accuracy and precision of the quantification of the target nucleic acid. In a standard curve, efficiency is typically calculated based on the slope of the curve, with higher efficiency resulting in steeper slopes. The efficiency of a qPCR reaction can be influenced by a number of factors, including the quality of the primers, the temperature and cycling conditions, and the characteristics of the target nucleic acid.
In an absolute standard curve, the goal is to determine the exact concentration of the target nucleic acid in a sample based on a known standard curve. In this case, the efficiency of the qPCR reaction is less critical than in a relative standard curve, where the goal is to compare the expression or abundance of the target nucleic acid in different samples.
While it is true that efficiency is less critical in an absolute standard curve, it is still important to optimize the efficiency of the reaction to ensure accurate and precise quantification. A low efficiency can result in an underestimation of the target nucleic acid concentration, which could lead to erroneous conclusions.
It is also worth noting that the efficiency of a qPCR reaction can vary between different qPCR machines and platforms, due to differences in the design and performance of the instruments. It is therefore important to validate the efficiency of the reaction on each instrument and platform that is used for the analysis.
The above answer is absolutely true, just to add few points
Sometimes it happens that the Instrument software follows different calculation for efficiency and one can always try to calculate manually.
I myself have used absolute quantification to calculate the efficiency as recommended by the MIQE guidelines and then perform the relative quantification.
The efficiency curve aids in understanding the efficiency of our primers to detect at minimal concentration
If you want to publish, then yes you'll need 95-105% efficiency. Read through the MIQE guidelines too.
https://pubmed.ncbi.nlm.nih.gov/19246619/
- Badges
- Science method