LRE modeling of real-time PCR. (A) The three inlaid equations allow PCR amplification to be modeled using ΔE and E
values derived from LRE analysis of cycles within the central region of each respective amplification profile (large red circles that correspond to the red circles in Figure 3). Following LRE analysis, each respective fluorescence reading (FC) is converted into target quantity (F0) using the second equation, from which an average F0 is calculated for each respective profile from the cycles included in the LRE analysis (Av. Fo). Reaction fluorescence can then be predicted using the third equation (black circles) that correlate closely to the actual fluorescence readings (dots). (B) Tabular summaries of the actual and predicted (Pred'd) reaction fluorescence for the cycles included in the LRE analysis, for each of the five amplification profiles. Percent difference (%Diff) illustrates the extraordinary precision that can be achieved, producing an average difference of < ± 0.2%. CV: coefficient of variation = standard deviation/average ×100%.