Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: microalgal pcna as the model gene

Abstract

Quantitative real-time PCR (qPCR) has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear) used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form) and linear DNA standards (linearized plasmid DNA or PCR amplicons), using proliferating cell nuclear gene (pcna), the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.

Figure 1. Standard curves with similar slopes and significant threshold cycle differences (ΔCt) between circular and linear standards in the pcna qPCR for (a) Alexandrium fundyense, (b) Karlodinium veneficum, (c) Prorocentrum micans, (d) P. minimum, and (e) Thalassiosira pseudonana.

Standard curves were linear regression lines between Ct and Log₁₀ starting pcna copy number (calculated from standard DNA concentration), each based on a type of standard DNA. Note that similar slopes of the standard curves indicate similar amplification efficiencies. All ΔCt were calculated as the average Ct difference across serial dilutions and statistically significant (p<0.001). The error bars denote the standard deviations of Ct values among replicates.

Figure 2. Comparison of qPCR-estimated and expected pcna copy numbers in Thalassiosira pseudonana gDNA samples.

The expected copy numbers were calculated based on 1 pcna per genome (0.035 pg of gDNA). Note that the copy number estimates based on the linear standard (TpsL) are similar with the expected numbers, while those based on the circular standard (TpsC) are much higher than the expected values. The error bars denote the standard deviations.