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Updated: Sep 21, 2025
A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications
Published on: September 16, 2022
Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and
Nicole Laurencia Yuwono1, Mollie Ailie Acheson Boyd1, Claire Elizabeth Henry1
1Gynaecological Cancer Research Group, Adult Cancer Program, School of Women's and Children's Health, Faculty of Medicine and Health, Lowy Cancer Research Centre, University of New South Wales, Sydney, Australia.
Insights
Storing circulating DNA (cirDNA) in plasma or as purified DNA for up to two years significantly reduces yield, with shorter fragments degrading faster. This impacts long-term study designs.
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Circulating DNA (cirDNA) analysis is vital for diagnostics and research.
- Biobanking of plasma for cirDNA extraction can span several years.
- Understanding cirDNA stability during storage is crucial for reliable results.
Purpose of the Study:
- To assess the impact of up to two years of frozen storage on cirDNA yield and fragmentation.
- To determine the stability of cirDNA when stored as plasma versus purified DNA.
Main Methods:
- Frozen EDTA plasma and purified cirDNA from 10 donors were stored for up to 2 years.
- cirDNA yield was quantified using qPCR and Qubit at regular intervals.
- Haemolysis and DNA extraction parameters were also evaluated.
Main Results:
- Annual cirDNA yield declined by 25.5% when stored as plasma and 23% as purified DNA.
- Shorter cirDNA fragments were degraded more rapidly than longer fragments.
- Plasma input and elution volume affected yield, but haemolysis did not.
Conclusions:
- Long-term storage of plasma and purified cirDNA leads to significant yield loss and fragmentation.
- Study designs for long-term cirDNA analysis must account for storage-induced degradation.
- Sample quality assurance is essential for reproducible cirDNA research.
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.
