Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma
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Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnostics, and prediction and monitoring of treatment response, but its amount is usually limited. Therefore, the choice of methods to extract and characterize ccfDNA is crucial. In the current study, we performed the most comprehensive comparison of methods for ccfDNA extraction (11 methods), quantification (3 methods), and estimation of the integrity index (2 methods) from small quantities of different kinds of plasma. The QIAamp® Circulating Nucleic Acid Kit and the Norgen Plasma/Serum Circulating DNA Purification Mini Kit showed the best accuracy and reproducibility, but the Norgen kit allowed to extract a higher amount of ccfDNA. This workflow provides a reliable protocol for the multiple applications of ccfDNA in biomedicine.
KeywordsCirculating cell-free DNA Plasma Extraction Quantification Integrity index Cancer Non-invasive diagnostics and personalized treatment
The authors would like to thank Chao Feng (CNG) for his help with the statistical analysis and Steven McGinn (CNG) for the critical reading of the manuscript. This work was partly supported by the EU FP7 under grant agreement number 241669 (the CAGEKID project, http://www.cng.fr/cagekid).
- 1.Esposito A, Bardelli A, Criscitiello C, Colombo N, Gelao L, Fumagalli L, Minchella I, Locatelli M, Goldhirsch A, Curigliano G (2014) Monitoring tumor-derived cell-free DNA in patients with solid tumors: clinical perspectives and research opportunities. Cancer Treat Rev 40:648–655CrossRefGoogle Scholar
- 2.Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, Parkinson C, Chin SF, Kingsbury Z, Wong AS, Marass F, Humphray S, Hadfield J, Bentley D, Chin TM, Brenton JD, Caldas C, Rosenfeld N (2013) Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497:108–112CrossRefGoogle Scholar
- 6.Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochim Biophys Acta 1775:181–232Google Scholar
- 15.Hufnagl C, Stöcher M, Moik M, Geisberger R, Greil R (2013) A modified phenol-chloroform extraction method for isolating circulating cell free DNA of tumor patients. J Nucleic Acids Investig 4:1Google Scholar
- 17.Thierry AR, Mouliere F, El Messaoudi S, Mollevi C, Lopez-Crapez E, Rolet F, Gillet B, Gongora C, Dechelotte P, Robert B, Del Rio M, Lamy PJ, Bibeau F, Nouaille M, Loriot V, Jarrousse AS, Molina F, Mathonnet M, Pezet D, Ychou M (2014) Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med 20:430–435CrossRefGoogle Scholar
- 18.Lebofsky R, Decraene C, Bernard V, Kamal M, Blin A, Leroy Q, Rio Frio T, Pierron G, Callens C, Bieche I, Saliou A, Madic J, Rouleau E, Bidard FC, Lantz O, Stern MH, Le Tourneau C, Pierga JY (2015) Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types. Mol Oncol 9:783–90CrossRefGoogle Scholar