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. 2014 Mar 3;9(3):e87838.
doi: 10.1371/journal.pone.0087838. eCollection 2014.

Direct quantification of cell-free, circulating DNA from unpurified plasma

Sarah Breitbach  1 , Suzan Tug  1 , Susanne Helmig  1 , Daniela Zahn  2 , Thomas Kubiak  2 , Matthias Michal  3 , Tommaso Gori  4 , Tobias Ehlert  1 , Thomas Beiter  5 , Perikles Simon  1
Affiliations

Direct quantification of cell-free, circulating DNA from unpurified plasma

Sarah Breitbach et al. PLoS One. .

Abstract

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Linearity of the L1PA2 qPCR.
A. Linearity in the standard curve of L1PA2 906 to 100 copies per reaction in TE and in mixtures spiked with murine plasma, to meet the characteristics of the direct measurement of cfDNA in human plasma. The qPCR efficiency was not affected by the ingredients of murine plasma. B. Dilution series of a human plasma cfDNA sample: the correlation of calculated and measured concentrations (upper: L1PA2 90 bp amplicon; lower: L1PA2 222 bp amplicon) shows a slight drift as a result of the qPCR efficiency of 93.97% that was applied for the calculations of absolute concentrations (solid line = regression line; dashed line = angle bisector).
Figure 2
Figure 2. Comparison of the qPCR results from the multilocus L1PA2 and the single-locus MSTN sequence amplification.
A. Absolute cfDNA concentrations measured with the L1PA2 and the MSTN qPCR in the eluate of DNA purification with the QIAamp DNA Blood Mini Kit (mean concentrations and standard deviations), showing a good concordance between amplifications of single and multi-locus repeats. B. Correlation of cfDNA concentrations (mean triplicate values). C. Paired differences for mean concentrations measured for the L1PA2 and the MSTN repeats.
Figure 3
Figure 3. Comparison of cfDNA concentrations measured in unpurified and purified plasma.
A. Absolute cfDNA concentrations post exercise measured with the L1PA2 qPCR in plasma and in the eluate and flow-through of the QIAamp DNA Blood Mini Kit. The concentrations determined in the flow-through were summed up with the concentrations in the eluate (sumFBM). B. Fold-difference between the absolute concentrations measured directly in plasma compared to the eluate of the QIAamp DNA Blood Mini Kit and to the sumFBM. C. Scatter plot showing the Spearman's correlation of cfDNA concentrations in plasma and the sumFBM (solid line = regression line; dashed line = angle bisector).
Figure 4
Figure 4. DNA integrity in unpurified and purified plasma samples.
Paired differences of DNA integrity values measured directly in plasma and in the eluate of the QIAamp DNA Blood Mini Kit.
Figure 5
Figure 5. Data from the Fragment Analyzer™ measurement shown by an example of one subject.
A. Sampling pre exercise and DNA extraction with the QIAamp DNA Blood Mini Kit; B. Sampling pre exercise and DNA extraction with PCI procedure; C. Sampling post exercise and DNA extraction with the QIAamp DNA Blood Mini Kit; D. Sampling pre exercise and DNA extraction with PCI procedure (LM = lower marker (35 bp); UM = upper marker (1500 bp)).
Figure 6
Figure 6. Overlay graphic of Fragment Analyzer™ data from 5 subjects pre and post exercise.
The samples presented here were purified using the QIAamp DNA Blood Mini Kit. At both time points, pre and post exercise, all samples showed a peak at approximately 170 bp. Post exercise, the concentrations of the this fragment length increased and a second peak was evident at on average 360 bp of length.
Figure 7
Figure 7. Comparison of cfDNA concentrations measured in 74 CHD patients and 74 healthy controls.
cfDNA concentrations were significantly higher in the patient collective. The data were presented as absolute cfDNA concentrations (ng/ml), while logarithmic data were inserted for the comparison of means.
See this image and copyright information in PMC

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