Next Article in Journal
The Human Microbiota and Prostate Cancer: Friend or Foe?
Previous Article in Journal
CX Chemokine Receptor 7 Contributes to Survival of KRAS-Mutant Non-Small Cell Lung Cancer upon Loss of Epidermal Growth Factor Receptor
Previous Article in Special Issue
A Pilot Clinical Study on the Prognostic Relevance of Plasmatic Exosomes Levels in Oral Squamous Cell Carcinoma Patients
Article Menu

Export Article

Open AccessArticle
Cancers 2019, 11(4), 458;

Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol

Center for Oncological Research Antwerp (CORE), University of Antwerp (UAntwerp), 2610 Wilrijk, Belgium
Laboratory of Pathological Anatomy, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
Biobank UZA/UAntwerpen, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
Biomedical Quality Assurance Research Unit, KU Leuven, 3000 Leuven, Belgium
Hepatobiliary Transplantation and Endocrine Surgery, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
Department of Oncology, Multidisciplinary Oncological Center Antwerp, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
Department of Pulmonology and Thoracic Oncology, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
Thoracic Medical Oncology and Early Clinical Trials, Marlene and Steward Greenebaum Comprehensive Cancer Center, Baltimore, MD 21201, USA
Author to whom correspondence should be addressed.
Received: 23 January 2019 / Revised: 15 March 2019 / Accepted: 27 March 2019 / Published: 30 March 2019
(This article belongs to the Special Issue Liquid Biopsy for Cancer)
PDF [2124 KB, uploaded 30 March 2019]
  |     |  


The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K2EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis. View Full-Text
Keywords: liquid biopsy; cell-free DNA; cell-free RNA; centrifugation protocol; ddPCR liquid biopsy; cell-free DNA; cell-free RNA; centrifugation protocol; ddPCR

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

Supplementary material


Share & Cite This Article

MDPI and ACS Style

Sorber, L.; Zwaenepoel, K.; Jacobs, J.; De Winne, K.; Goethals, S.; Reclusa, P.; Van Casteren, K.; Augustus, E.; Lardon, F.; Roeyen, G.; Peeters, M.; Van Meerbeeck, J.; Rolfo, C.; Pauwels, P. Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol. Cancers 2019, 11, 458.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics



[Return to top]
Cancers EISSN 2072-6694 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top