Abstract 486: A comparison of cfDNA isolation kits: isolation and quantification of cell-free DNA in plasma
Authors: Sorber Laure , Zwaenepoel Karen , Deschoolmeester Vanessa , Roeyen Geert , Goethals Sofie , Rolfo Christian , Pauwels Patrick 
Published: July 2016

Introduction: In oncology, circulating cell-free DNA (cfDNA) has emerged as a new and sensitive biomarker. However, the low concentrations of cfDNA, especially in early stages of disease, make it a challenging analyte for extraction, consequently high isolation efficiency is essential.

In this study we compared the isolation efficiency of one of the most commonly used DNA isolation kits, namely the QIAamp® circulating nucleic acid kit (QIA - Qiagen), with four newly launched cfDNA isolation kits.

Material & Methods: The QIA kit was compared with the PME free-circulating DNA Extraction Kit (PME - Analytik Jenna), the Maxwell® RSC ccfDNA Plasma Kit (RSC - Promega), the NEXTprep-MagTM cfDNA Isolation Kit (NpM - BiooScientific), and the EpiQuickTM Circulating Cell-Free DNA Isolation Kit (EQ - Epigentek).

CfDNA was extracted from 10 plasma samples of patients treated at the Antwerp University Hospital (UZA); including two patients with benign pancreatic cysts, three early stage pancreatic cancer patients, and four pancreatic cancer patients with metastasized disease, of which one had a second blood sample taken during follow up. Only the patients with metastasized disease were KRAS mutated.

To determine the isolation efficiency of the different kits, the QX200TM Droplet DigitalTM PCR system (ddPCR - Bio Rad) was used. All samples were screened with the ddPCRTM KRAS Screening Multiplex kit (Bio Rad) for the presence of seven KRAS mutations and a specific wild type sequence, which results in the KRAS mutated cfDNA fraction and the quantification of the total cfDNA.

Results: The QIA and the RSC kits displayed similar isolation efficiencies of both KRAS mutated and non-mutated cfDNA; with the detection of the KRAS mutated fraction ranging from approximately 1% to 40% and on average a yield of 76,7 and 83,1 copies/μl, respectively. On the other hand, the PME kit could not detect any KRAS mutations and in four cases there was not enough cfDNA isolated to be detected with ddPCR. DdPCR of cfDNA isolated with the NpM and EQ kits did not generate any positive signals. qPCR was performed on cfDNA isolated with both kits with control primers of the NpM kit. All samples of the NpM kit and three of the samples of the EQ kit were shown to contain cfDNA of at least 180 bp; which leads to the conclusion that ddPCR can't be used as a downstream procedure for the NpM and EQ kits.

Conclusion: The RSC kit enables the isolation of a sufficient quantity of cfDNA similar to the yield generated with the QIA kit, and both kits have a higher isolation efficiency than the PME kit. This study presents two highly efficient isolation kits, of which the RSC kit has the advantage of a fully automated, magnetic beads based protocol over the labor intensive and time consuming QIA kit. We also concluded that ddPCR as a downstream procedure is not feasible with the NpM and EQ kits. However, an improved version of the NpM kit has recently been produced and will also be included in this comparison study.

Citation Format: Laure Sorber, Karen Zwaenepoel, Vanessa Deschoolmeester, Geert Roeyen, Sofie Goethals, Christian Rolfo, Patrick Pauwels. A comparison of cfDNA isolation kits: isolation and quantification of cell-free DNA in plasma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 486.