Comparison of absolute quantification of DNA/RNA in complex biological samples by qPCR and Crystal dPCR

Comparison of the effects of inhibitors on quantitative detection of DNA/RNA by dPCR and qPCR ( Real-time PCR )

It is well known that thousands of chemicals inhibit the PCR reaction, and biological samples often contain similar compounds, which are difficult to remove during DNA/RNA purification and are therefore present in DNA/RNA samples.

> q PCR and C rystal dPCR for DNA /RNA quantification comparison

When qPCR (Real-time PCR) is used for DNA/RNA quantification, based on the Ct value and depending on the standard curve between the sample and the standard analyzed, the Ct value between the unknown sample and the known nucleic acid standard is obtained. The concentration of the unknown sample. However, when an inhibitor is present in the sample, the PCR reaction efficiency may be different between the standard and the sample due to the background, so the quantitative results will be biased (Fig. 1A).

When Naica crystal dPCR was used for DNA/RNA quantification, the results were judged by the positive or negative differentiation of droplets by fluorescence values. Even if there is a decrease in the efficiency of the PCR reaction due to the presence of the inhibitor, accurate quantification can be achieved (Fig. 1B).

Figure 1. Comparison of the effects of humic acid in qPCR(A) and Crystal dPCR(B) systems

The PCR reactions in both qPCR and Crystal dPCR platform assays included 1X PerfeCta Multiplex qPCR Tough-mix, 100 nM fluorescein, 500 nM ALB gene forward and reverse primers, 250 nM Cy5 labeled TaqMan probe and 5.4 ng / uL human genome DNA. The humic acid was mixed at 0, 50 and 100 ug / mL, respectively.

It is also important to note that inhibitors may have different effects on the absolute quantification of different target genes in the same DNA/RNA sample (Figure 2). The Naica Crystal dPCR system has three fluorescent channels that detect different target genes in different regions or genomes of the gene of interest to examine this bias.

Figure 2. Detection of the effect of humic acid on different targets in Crystal dPCR

Multiple Crystal DPCR reactions containing PerfeCta Multiplex qPCR Toughmix, primers and probes for the ALB gene (Cy-5 tag), primers and probes for the EGFR gene (HEX tag) and primers and probes for BRAF (FAM tag), and used The same amount of human genomic DNA was spiked with different concentrations of humic acid (fitted with a Sigmoid or S-type function). The percentage of inhibition was calculated by quantification of DNA quantification without addition of inhibitor.

> Comparison of resistance of qPCR and Crystal dPCR to inhibitors

We compared the effects of two known inhibitors found in the sample on DNA quantification on qPCR and Crystal dPCR platforms, namely the presence of humic acid in soil samples and the use of anticoagulation in collected blood samples. Heparin. The results show that Crystal dPCR can still be accurately quantified in the presence of any higher concentration of inhibitor compared to qPCR (Figure 3).

Figure 3. Inhibition of qPCR and Crystal dPCR by different concentrations of humic acid (A) and heparin (B).

The qPCR and Crystal dPCR reactions contained PerfeCta Multiplex qPCR Toughmix, the same amount of human genomic DNA, and primers and probes that target the BRAF gene for humic acid inhibition and the EGRF gene for heparin inhibition. All samples were subjected to 2 experiments with three replicates per experiment (fitting with Sigmoid or sigmoid function). The percentage of inhibition was calculated by quantification of DNA quantification without addition of inhibitor.

In summary, Crystal dPCR can be more reliable than qPCR results when performing DNA/RNA analysis on samples with inhibitors. Crystal dPCR is the best solution you've been looking for for nucleic acid detection and quantification in samples containing inhibitors.