Abstract
Objectives
Circulating DNA (cirDNA) is generally purified from plasma that has been biobanked for variable lengths of time. In long-term experiments or clinical trials, the plasma can be stored frozen for up to several years. Therefore, it is crucial to determine the stability of cirDNA to ensure confidence in sample quality upon analysis. Our main objective was to determine the effect of storage for up to 2 years on cirDNA yield and fragmentation.
Methods
We stored frozen EDTA plasma and purified cirDNA from 10 healthy female donors, then quantified cirDNA yield at baseline, and at regular intervals for up to 2 years, by qPCR and Qubit. We also compared cirDNA levels in non-haemolysed and haemolysed blood samples after 16 months of storage and tested the effect of varying DNA extraction protocol parameters.
Results
Storage up to two years caused an annual cirDNA yield decline of 25.5% when stored as plasma and 23% when stored as purified DNA, with short fragments lost more rapidly than long fragments. Additionally, cirDNA yield was impacted by plasma input and cirDNA elution volumes, but not by haemolysis.
Conclusions
The design of long-term cirDNA-based studies and clinical trials should factor in the deterioration of cirDNA during storage.
Funding source: Beth Yarrow Memorial Award in Medical Science
Award Identifier / Grant number: NA
Funding source: CAMILLA AND MARC
Award Identifier / Grant number: NA
Funding source: Gynaecological Oncology Fund
Award Identifier / Grant number: NA
Funding source: Translational Cancer Research Network
Award Identifier / Grant number: NA
Funding source: Australian Research Training Program
Award Identifier / Grant number: NA
Funding source: Ovarian Cancer Research Foundation
Award Identifier / Grant number: GA-2018-14
Acknowledgments
We would like to acknowledge and thank the volunteers for their blood donation. We also thank Professor Susan Ramus for her advice on study design as well as Dr. Kylie-Ann Mallitt and Dr. Nancy Briggs for statistics consultation.
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Research funding: Nicole Yuwono and Bonnie Werner was supported by the Australian Government Research Training Program (RTP) Stipend through The University of New South Wales and Translational Cancer Research Network PhD Scholarship Top Up Award, via the Cancer Institute NSW. Nicole Yuwono was further supported by Beth Yarrow Memorial Award in Medical Science and UNSW Completion Scholarship. Claire Henry was supported by the Gynaecological Oncology Fund of the Royal Hospital for Women, Sydney. Dr. Kristina Warton was supported by Ovarian Cancer Research Foundation (GA-2018-14) and CAMILLA AND MARC. The funders had no role in the decision to publish or preparation of the manuscript.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Dr. Kristina Warton holds stock in Guardant Health, Exact Sciences and Epigenomics AG. No other authors have competing interests.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the University of New South Wales Human Research Ethics Committee (#HC17020).
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2021-1152).
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