qPCR Amplification Efficiency too high - Efficiency of 115 - 145% (Aug/18/2010 )
I recently tested amplification efficiency on several genes using Taqman primers and probes. I created 5 10x dilutions, from 10 ng/uL down to 0.001 ng/uL cDNA (total RNA equivalents). I added 2 uL of each dilution of cDNA to the reaction well, using a 10 uL total reaction volume (ABI said 10 uL is fine for use and I validated using 10 uL uniplex). Half of the primer/probes are 2 1/2 years old, while the other half are 2 months old.
Half of the old combos and half of the new ones had amplification efficiencies of 115 - 145%. The r^2 value was between 0.99 and 1.00. The amplification plots look normal. The difference in slope between the housekeeping genes and genes of interest is greater than 0.1. I have a spreadsheet set up, so I can easily adjust the relative abundance based on the emperical amplification efficiency values.
I saw another topic just like this one, but it wouldn't let me post, so I've addressed their concerns here. PCR inhibitors decrease efficiency, they don't increase it, so I don't have issues with inhibitors. The age of the probes is not really my first thought as some of the 2 month old probes are also displaying high E values and some of the old combos have E values of 1.97 - 2.02.
Any help would be much appreciated. Thanks.
Hi Anne86,
We have recently developped a new method to overcome significant quantitative inaccuracy due to slight amplification inhibition. I don't know if this fits with your need, but if you want feel free to see furhter detail at cy0method
Best Regard Jazzland
Anne86 on Wed Aug 18 20:16:48 2010 said:
I recently tested amplification efficiency on several genes using Taqman primers and probes. I created 5 10x dilutions, from 10 ng/uL down to 0.001 ng/uL cDNA (total RNA equivalents). I added 2 uL of each dilution of cDNA to the reaction well, using a 10 uL total reaction volume (ABI said 10 uL is fine for use and I validated using 10 uL uniplex). Half of the primer/probes are 2 1/2 years old, while the other half are 2 months old.
Half of the old combos and half of the new ones had amplification efficiencies of 115 - 145%. The r^2 value was between 0.99 and 1.00. The amplification plots look normal. The difference in slope between the housekeeping genes and genes of interest is greater than 0.1. I have a spreadsheet set up, so I can easily adjust the relative abundance based on the emperical amplification efficiency values.
I saw another topic just like this one, but it wouldn't let me post, so I've addressed their concerns here. PCR inhibitors decrease efficiency, they don't increase it, so I don't have issues with inhibitors. The age of the probes is not really my first thought as some of the 2 month old probes are also displaying high E values and some of the old combos have E values of 1.97 - 2.02.
Any help would be much appreciated. Thanks.