Effect of difference in efficiency between standard samples and samples. This figure illustrates the error in determination of concentration by real-time PCR at cycle no. 30 when the efficiencies differ between standard samples and samples. Number of cycles: n, Efficiency standard samples: Estd, efficiency reference samples: Ers, Number of amplicons at cycle n: Nn, Number of amplicons at cycle zero: N0. When the two equations are divided by eachother it is seen that there is an 23-fold underestimation of the concentration when Estd = 2.0 and Ers = 1.8. From this calculation it is also evident that at higher cycle number the error in concentration determination is larger.