Comparing tolerance of qPCR and Crystal Digital PCR™ to inhibitors
We compared the effects of two known inhibitors, humic acid, found in soil samples, and heparin, used as an anticoagulant in collected blood samples, on DNA quantification both in real-time PCR and Crystal Digital PCR. We found that accurate quantification remains possible in presence of higher concentrations of either inhibitor when using Crystal Digital PCR, compared to real-time PCR (Figure 3).
Figure 3. Concentration-dependent inhibitory effect of humic acid (A) and heparin (B) on real-time PCR (qPCR) and Crystal Digital PCR. PCR reactions for either qPCR or Crystal digital PCR contained PerfeCta Multiplex qPCR Toughmix, identical quantities of human genomic DNA, as well as primers and probes targeting either the BRAF gene when testing for humic acid or EGFR gene when testing for heparin. All samples were assayed in triplicates for N=2 experiments (plotted with sigmoid fit). Percentage of inhibition is calculated considering 0% inhibition for DNA quantification with no inhibitor.
In conclusion, when dealing with inhibition-prone samples, digital PCR can be more reliable than qPCR.
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