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Letters to the Editor |
1 Charite–Universitätsmedizin Berlin CCM
2 Medizinische Klinik mit Schwerpunkt, Kardiologie, Pulmologie und Angiologie, und
3 Medizinische Klinik mit Schwerpunkt, Onkologie und Hämatologie, Berlin, Germany
aAddress correspondence to this author at: Charite–Universitätsmedizin Berlin, CCM, Medizinische Klinik mit Schwerpunkt Onkologie und Hämatologie, Schumannstrasse 20-21, 10117 Berlin, Germany. Fax 49-30-450-51-39-64; e-mail michael.fleischhacker{at}charite.de.
To the Editor:
After Mandel and Metais (1) had originally described the existence of free circulating nucleic acids in plasma/serum in humans, Stroun et al. (2)(3) demonstrated that part of the free circulating DNA in cancer patients is of tumor origin (4)(5)(6).
The quantity of DNA that can be isolated from human plasma/serum is very low and is frequently the limiting factor when a larger marker panel is to be used for genetic characterization of the DNA. In most of the recently published studies, commercially available columns were used for isolation of cell-free plasma/serum DNA, which made DNA isolation fast and allowed it to be standardized. Mandel and Metais (1) found 
5.4 µg/mL total nucleic acids in the plasma in 10 healthy controls, and Stroun et al. (3) isolated between 150 ng/mL and 12 µg/mL DNA from the plasma of cancer patients. Much less DNA can be isolated with the aforementioned columns, however; we therefore wondered whether there are ways to increase the yield of free circulating DNA that can be isolated from plasma/serum and other body fluids. We compared the DNA yield from 1 mL of material, using 2 methods. Plasma and cell-free bronchial lavage supernatants (BL) were prepared as described (7). For the first method, we used the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. For the second method, we modified the DNA method described by Miller et al. (8).
To 1 mL of starting material, we added 100 µL of a solution containing 250 mmol/L EDTA and 750 mmol/L NaCl and 100 µL of 100 g/L sodium dodecyl sulfate and proteinase K (final concentration, 100 mg/L). The samples were incubated overnight at 50 °C, and the proteins were precipitated with 300 µL of saturated NaCl solution (final concentration, 1.2 mol/L), vortex-mixed, and centrifuged for 15 min at 6000g. The cleared supernatant was transferred into a new tube, and the DNA was precipitated by adding the same volume of absolute ethanol and incubating overnight at –20 °C. The DNA was centrifuged for 30 min at 16 440g, dissolved in water, extracted once with a 1:1 phenol–chloroform mixture, extracted a second time with chloroform only, and then ethanol-precipitated at –20 °C. Finally, the DNA pellets were centrifuged, washed with 750 mL/L ethanol, air-dried, and dissolved in 50 µL of 10 mmol/L Tris–1 mmol/L EDTA. We used 3 µL of template DNA for quantification, and each sample was analyzed in duplicate. The DNA was quantified by a real-time PCR analysis using the sequence of ERV-3, a human endogenous retrovirus, as target (9). Details for the TaqMan assay are supplied in the Data Supplement that accompanies the online version of this Letter at http://www.clinchem.org/content/vol51/issue8/ (10).
The DNA yield obtained with our new method from cell-free BL from tumor patients and controls was higher than the amount obtained with the Qiagen columns (P = 0.05 and 0.03; respectively; Table 1
). A possible explanation for the lower yield with the columns might be that these columns are effective in binding nucleic acid molecules larger than 100–150 bp (information obtained from Qiagen Technical Service). To determine the recovery rate for the columns, the DNA from 13 BL samples, which had initially been isolated by the salting-out method, were "repurified" with the Qiagen columns and again quantified. Approximately 50% of the DNA was tightly bound to the column and was not eluted even when the elution step was done twice and the columns were incubated with preheated elution buffer for 1–5 min (recommended by the manufacturer). These results are in keeping with another report, in which the yield of plasma DNA obtained by the QIAamp DNA Blood Mini Kit ranged from 40% to 60% (11).
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Our observation that the amount of DNA obtained from BL is much higher than the amount obtained from plasma confirms earlier results (our unpublished data) and underlines the potential value of the examination of cell-free nucleic acids obtained from BL (7). Our results also corroborate the conclusion made by others that the diagnostic value of the plasma DNA concentration is low (11). This contradicts a report in which the authors found a substantial difference between the amounts free circulating DNA in the plasma of lung tumor patients and patients without lung tumors (12).
In summary, it is possible to increase the amount of free circulating DNA that can be isolated from different body fluids up to 8-fold by a modified salting-out protocol. The fact that this method is more time-consuming than the use of columns is easily compensated for by the higher yield of DNA.
Acknowledgments
This work was supported by a grant from the Monika Kutzner Stiftung to Drs. Fleischhacker and Schmidt. We would like to thank K. Jung for helpful comments.
References
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