Efficiency>2! What does this mean? - (Apr/20/2006 )
I am setting up a PCR and performed 10 fold serial dilutions to obtain a graph of Cp vs log copy number. The slope of this graph is -3.0398 which using the equation E=10^-1/slope=2.14. I thought that 2 was the maximum. What does it mean to have an efficiency>2?
An efficiency > 2 can mean several things..and here are a couple that come to mind:
1. Pipetting error
2. Inhibition..As any inhibitors present in your cDNA are diluted out, the efficiency of amplification improves as compared with the lower dilutions, leading to a perceived higher than 100% efficiency of the assay.
Also, if the Ct values of your highest dilutions are in the high 30s, those values are not as reliable as you are getting into single copies of template. You might try analyzing your data while dropping out values over 35 (these could be out of your dynamic range) and see if that improves your efficiency.
if efficiency more than 2 it might be due to threshold level sets at the non linear phase. Make sure you already perform the slope correction/ dynamic normalization etc. to eliminate all the noise signals. By the way, I am using Rotor gene and these are the precaution we should do before taking the final Ct values. You can visit www.qiagen.com and have a look at the real time PCR notes/handbook, which you can downloaded in pdf file.