Absolute Quantification of Estimated Copy Number Without a Standard Curve

Absolute Quantification of Estimated Copy Number Without a Standard Curve

Abstract
The goal of any qPCR is to quantify DNA amounts by exponentially amplifying minute amounts to detectable levels. Usually, a certain fluorescence level is defined as the threshold and the number of cycles, needed by each reaction to reach this threshold, is reported as quantification cycle or Cq. However, the amplification efficiency and the level of the threshold have a large impact on the reported Cq. Therefore, comparing Cq values is only valid if the PCR efficiency is identical and the threshold is set at the same level for every assay. A common mistake is that these essential prerequisites are not ensured and thus biased data are reported.
A web based LinRegPCR was developed to determine the PCR efficiency from individual amplification curves. This program reports the N0 value, which is the calculated fluorescence of the target-of-interest at the start of the PCR. The N0 values are not dependent on the threshold and are corrected for differences in PCR efficiency. Therefore, they can easily be used in calculations of relative expression levels. However, because N0 values are based on the machine’s fluorescence scale, they differ between machines. Furthermore, these values are expressed as very small fractions of the fluorescence units and at first glance counterintuitive.
N0 values can be easily used for absolute quantification with a single standard. The copy number of all other reactions can be calculated by dividing the individual N0 values by the quantification factor which is the N0 value of the standard reaction divided by its known number of input copies. The advantage of using a single standard rather than a calibration curve is the avoidance of dilution errors.
Even without such a single standard or a calibration curve, estimated copy numbers can be calculated. To that end, a rule of thumb is applied: a reaction starting with 10 copies of the target will result in a Cq value of 35 cycles. Using this rule, one can calculate the quantification factor based on the threshold and assuming a PCR efficiency of 1.9 as well as an amplicon length of 100bp for this hypothetical standard. Because identical PCR reactions will result in amplification curves of similar shape, and thus a comparable threshold position, this approach will also compensate for differences between machines. Estimated copy numbers can thus be calculated for every qPCR reaction and provide an intuitive way of quantification of the number of target molecules in the amplification reaction. Furthermore, these estimated copy numbers correct for differences in efficiency and can be compared between different machines and laboratories.
All above described applications are part of the RDML-Tools, an open-source software (MIT & GPL) and server (https://www.gear-genomics.com/rdml-tools/). Furthermore, the RDML-Tools cover a complete qPCR analysis pipeline from the raw fluorescence data up to statistical evaluation of differences between groups.