Overview of LRE-based real-time qPCR.

<p>As described in detail in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009731#pone.0009731-Rutledge2" target="_blank">[2]</a>, LRE originated from recognition that amplification efficiency is linearly coupled to amplicon quantity and is made up of three broad components. <b>LRE analysis</b> is implemented by first estimating cycle efficiency (E_C) from the relative increase in reaction fluorescence over each individual thermocycle. E_C is then plotted against reaction fluorescence (F_C) to produce the LRE plot. In addition to generating a linear representation of PCR amplification, linear regression analysis of the cycles within the central region of the profile (denoted by red circles and referred to as the “LRE window”) provides values for two kinetic parameters, the maximal amplification efficiency, E_max (Y intercept) and the rate of loss in amplification efficiency, ΔE (slope). <b>Target quantification</b> involves conversion of the F_C readings within the LRE window into target quantity (F₀) using an equation derived from the classic Boltzmann sigmoid function. As described in a later section, the universal nature of SYBR Green I fluorescence allows the average F₀ to be converted into the number of target molecules using an optical calibration factor (OCF). <b>LRE modeling</b> utilizes a second sigmoid function to calculate predicted F_C values for each thermocycle (pFc). Comparison of predicted to actual F_C within the LRE window typically produces an average difference of <0.5%, indicative of the remarkable precision that can be achieved. Profile position can be defined by C_1/2, which is the fractional cycle at which reaction fluorescence reaches half of F_max.