Figure 4

Optimizing LRE window size via recursion. This data is taken from the 0.5X SYBR Green I DyNAmo amplification profile (Figure 2); however, all the amplification profiles in Figure 2 generate similar results. The recursive analysis starts by assigning a small LRE window (designated by red circles) in the lower region of the amplification profile and conducting linear regression analysis using the points within this preliminary LRE window (blue highlight). F_C readings are then converted to F₀ using equation 6 and the average determined for F₀ values encompassed by the LRE window (green highlight). Conformity of the F₀ value generated by the cycle immediate following the last cycle of the LRE window is then assessed (bold), based upon the percent difference with the LRE window F₀ average (%Diff, yellow highlight). If this difference is less than a specified threshold, the LRE window is expanded to include the next cycle and the analysis repeated. This recursive process is continued until a nonconforming cycle is encountered as defined by the threshold. Note that for illustrative purposes, the LRE window in this figure has been expanded beyond the threshold used in this study (7.5%). Although the LRE line generated within the LRE plot can be used to assess conformity, examining the resulting F₀ values provides a more sensitive and objective methodology, as is visually illustrated by the F₀ plots.