Purification of Circulating Cell-Free DNA from Plasma and Urine Using the Automated Large-Volume Extraction on the QIAsy
Increasing sample numbers for screening and diagnostics using circulating cell-free DNA (ccfDNA) as analyte demands an automated solution for ccfDNA extraction. The efficiency of a new, automated, large volume ccfDNA extraction method was evaluated agains
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Alexander Wolf, Katharina Beller, Sebastian Groemminger, Wera Hofmann, Matthias Sachse, and Jana Fassunke
Abstract
Increasing sample numbers for screening and diagnostics using circulating cell-free DNA (ccfDNA) as analyte demands an automated solution for ccfDNA extraction. The efficiency of a new, automated, large volume ccfDNA extraction method was evaluated against a manual reference method. The new kit for automated ccfDNA extraction on the QIAsymphony showed a comparable yield of total ccfDNA from healthy donors as well as a comparable recovery of circulating cancer and fetal DNA. In conclusion, a new kit for automated ccfDNA extraction was established successfully. Keywords
ccfDNA • Automation • Fetal • Cancer • QIAsymphony • QIAGEN
Introduction
A. Wolf (*) • K. Beller Research and Development, QIAGEN GmbH, 40724 Hilden, Germany e-mail: [email protected] S. Groemminger • W. Hofmann • M. Sachse LifeCodexx AG, 78467 Konstanz, Germany J. Fassunke Institute of Pathology, Universitätsklinikum Köln, 50924 Cologne, Germany
Circulating cell-free DNA (ccfDNA) is a key analyte for non-invasive prenatal diagnostics and cancer biomarker analysis. Due to extremely low concentrations and a high degree of fragmentation in sample material, the extraction of ccfDNA is technically challenging. Increasing sample numbers requires an automated extraction of ccfDNA. The automated solution must address a high sample input volume to compensate the low ccfDNA concentration while the DNA has to be extracted quantitatively from plasma and eluted
© Springer International Publishing Switzerland 2016 P.B. Gahan et al. (eds.), Circulating Nucleic Acids in Serum and Plasma – CNAPS IX, Advances in Experimental Medicine and Biology 924, DOI 10.1007/978-3-319-42044-8_33
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in a low elution volume to concentrate the DNA. In parallel, instrumentation and chemistry must maintain a high throughput. Here, the efficiency of a new, automated, large volume ccfDNA extraction method was evaluated against a manual reference method.
Materials and Methods Plasma was obtained by two-step centrifugation from K2-EDTA tubes and Cell-Free DNA (Streck) tubes. ccfDNA was extracted from either plasma or urine using QIAamp® Circulating NA Kit (QIAGEN, cat. no. 55114) as per the manual reference method and QIAsymphony® Circulating DNA Kit (QIAGEN, cat. no. 1091063) as per the automated extraction method. Urine required an ATL pre-treatment before ccfDNA extraction. ccfDNA yield was determined by the Qubit dsDNA HS Assay Kit (cat. no. Q32851) and real-time PCR (18S, forward primer: 5′-GCCGCTAGAGGTGAAATTCTTG-3′, reverse primer: 5′-CATTCTTGGCAAATGCTTTCG-3′, probe: 5′-ACCGGCGCAAGACGGACCAGA-3′) using the QIAGEN QuantiTect® Multiplex PCR Kit. Size distribution of ccfDNA was determined with the High Sensitivity DNA Kit and DNA 7500 Kit (Agilent). Cancer plasma samples: For the library preparation, an Ion AmpliSeqLibrary Kit 2.0, Thermo Fisher was used. Three nM of each ccfDNA was transferred to a pool that was diluted to 12 pM for targeted NGS.
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