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Comparative Study
. 2012 Jun;40(11):e82.
doi: 10.1093/nar/gks203. Epub 2012 Feb 28.

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation

Affiliations
Comparative Study

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation

Alexandra S Whale et al. Nucleic Acids Res. 2012 Jun.

Abstract

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

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Figures

Figure 1.
Figure 1.
Comparison of HER2:RNase P ratio in breast cancer cell line genomic DNA using digital and quantitative real-time PCR. (a) Software-generated heat map showing a single panel in a 48.770 dPCR array that contains 770 chambers with positive (white) and negative (black) amplification signals. One representative dPCR panel is shown for each gDNA sample and assay with the number of positive chambers shown in the top right corner of the panel. Positive and negative chambers were used to calculate the number of molecules per panel and the HER2:RNase P ratio for the gDNA sample. The NTC panels for both assays had no positive chambers. (b) qPCR (n = 8 wells) and dPCR (n = 4 panels) gave similar HER2:RNase P ratios for all BC gDNA except the SK-BR-3 gDNA that was significantly higher by qPCR compared with dPCR (asterisk). Data is presented on a log scale and error bars represent 95% CIs.
Figure 2.
Figure 2.
Determination of CNV detection of digital and quantitative real-time PCR. Quantitation of HER2:RNase P ratios using (a) dPCR and (b) qPCR generated from the in vitro gene-amplification model. The x-axis shows the expected HER2:RNase P ratio and the y-axis shows the observed HER2:RNase P ratios with the 95% CI. (a) For dPCR, four panels for each assay were analyzed for ratios > 1.5 (daggered symbol) and eight panels for each assay for ratios <1.5. In all cases, λr was approximately 0.2. The error bars represent the 95% CIs. (b) For qPCR, eight reactions were performed for each assay and all ratios. The error bars represent the 95% CIs calculated from the standard error of the mean and associated T-value with 95% confidence and seven degrees-of-freedom. Key: black triangle: normal female gDNA, black diamond: significantly different from normal female gDNA (P < 0.05), gray diamond: not significantly different from normal female gDNA (P > 0.05). Solid line of linear correlation is shown for those ratios that were significantly different from normal female gDNA. Dashed line is the extrapolation of the linear correlation showing intersection with HER2:RNase P ratio of 1.0. R2 and equations are given for the linear correlation.
Figure 3.
Figure 3.
Determination of the number of dPCR panels needed to measure HER2:RNase P ratios. (a) Power curve to determine the number of panels required to detect different ratios of λt to λr, where λt > λr with 95% power at a confidence level of 95% and λr = 0.2. The two horizontal lines show a single-panel and eight-panel experiment where the intersections with the power curve indicates the lowest detectable CNV. The vertical line shows the smallest CNV detectable is approximately 1.15 when λr = 0.2 and the number of panels is 8. (b) The relevant number of dPCR panels (1–8) were selected and the HER2:RNase P ratio and associated 95% CIs were calculated. The graphs for the different ratios are slightly staggered to allow identification of the 95% CI error bars for each ratio. Ratios that are statistically different from the normal female gDNA are shown for ratios of 1.27 (gray asterisk) and 1.17 (black asterisk).

References

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