Absolute quantification of references without a standard curve
Accurate genetic measurements often require comparison to reference samples and assay standards. Standardization of references is especially important in the field of metrology. For many organisms or applications, there is often no suitable reference sample available. Generation of reference standards using conventional real-time PCR requires consideration of how the reference sample will initially be calibrated, its long-term stability, and whether there is sufficient reference material for completion of all future studies. In addition, the lack of broadly adopted standards impacts comparison between laboratories.
Digital PCR does not rely on a reference sample or assay standard; it can be used for absolute quantification, measuring the exact copy number of a nucleic acid target of interest. This capability is especially useful for calibrating reference samples and assay standards when none exist. Through direct copy number determination, digitally measured assay standards can enable laboratories to compare results, with the assurance that measurements are based on the same absolute baseline.
Figure 1. Digital PCR precisely and accurately quantifies standards without the use of a standard curve. (A) Four standards were measured in duplicate, and results determined in absolute copies per microliter by digital PCR. The tight error bars demonstrate very high precision in the measurement of each sample. (B) For sample 600-T, an additional 10-fold dilution series covering four logs of dilution was constructed. Copies per reaction for each dilution were calculated and demonstrate excellent correlation (0.9991), with extremely tight precision for each dilution.
Measuring the absolute number of molecules in a biological sample is a relatively common need. But, this can pose some unique challenges when using standard real-time PCR. In contrast, digital PCR is not dependent on cycle thresholds or references, yielding a simple and precise method to obtain absolute quantification.
For Research Use Only. Not for use in diagnostic procedures.