Unlike end point PCR (conventional PCR), real time PCR allows monitoring of the desired product at any point in the amplification process by measuring fluorescence (in real time frame, measurement is made of its level over a given threshold). A commonly employed method of DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a logarithmic scale. A threshold for detection of DNA-based fluorescence is set 3–5 times of the standard deviation of the signal noise above background. The number of cycles at which the fluorescence exceeds the threshold is called the threshold cycle (C) or, according to the MIQE guidelines, quantification cycle (C).
During the exponential amplification phase, the quantity of the target DNA template (amplicon) doubles every cycle. For example, a DNA sample whose C precedes that of another sample by 3 cycles contained 2 = 8 times more template. However, the efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in (C) with each dilution. The slope of the linear regression is then used to determine the efficiency of amplification, which is 100% if a dilution of 1:2 results in a (C) difference of 1. The cycle threshold method makes several assumptions of reaction mechanism and has a reliance on data from low signal-to-noise regions of the amplification profile that can introduce substantial variance during the data analysis.
To quantify gene expression, the (C) for an RNA or DNA from the gene of interest is subtracted from the (C) of RNA/DNA from a housekeeping gene in the same sample to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the ΔC-method and permits comparison of expression of a gene of interest among different samples. However, for such comparison, expression of the normalizing reference gene needs to be very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues. Although cycle threshold analysis is integrated with many commercial software systems, there are more accurate and reliable methods of analysing amplification profile data that should be considered in cases where reproducibility is a concern.
Mechanism-based qPCR quantification methods have also been suggested, and have the advantage that they do not require a standard curve for quantification.  Methods such as MAK2 have been shown to have equal or better quantitative performance to standard curve methods.  These mechanism-based methods use knowledge about the polymerase amplification process to generate estimates of the original sample concentration. An extension of this approach includes an accurate model of the entire PCR reaction profile, which allows for the use of high signal-to-noise data and the ability to validate data quality prior to analysis.
According to research of Ruijter et al. MAK2 assumes constant amplification efficiency during the PCR reaction. However, theoretical analysis of polymerase chain reaction, from which MAK2 was derived, has revealed that amplification efficiency is not constant throughout PCR. While MAK2 quantification provides reliable estimates of target DNA concentration in a sample under normal qPCR conditions, MAK2 does not reliably quantify target concentration for qPCR assays with competimeters.