Figure 1. DNA concentration measurements by Qubit (left axis) and qPCR (CCR5 amplification) (right axis) on cfDNA extracted from plasma collected in Nonacus Cell3™ Preserver and Streck tubes and isolated at 3, 6, 13 and 20 days post blood draw. Streck tubes show an apparent increase in cfDNA concentration from day 13 due to gDNA released by the lysis of contaminating white blood cells.
As there is only a low concentration of cfDNA in plasma, it is important as well as minimising gDNA contamination, to start with adequate volumes of whole blood. In fact the predominant challenge for molecular diagnostics in both oncology and prenatal genetics is that an even smaller and variable fraction of cfDNA – potentially as little as 1% -is derived from either the tumour (circulating tumor DNA (ctDNA)) or the foetus (circulating free foetal DNA (cffDNA)).
To enable detection of low frequency variants by NGS in either of these examples, a sample volume of 8mL of whole blood is recommended as a starting amount. Approximately half of that volume, 4mL, should be recovered as plasma, so you may expect, in a healthy individual, to recover 1-5ng of cfDNA per mL of plasma.
Plasma collection and storage
If you obtain 4mL plasma per 8ml tube of whole blood, then aim to use the whole sample for extraction. It is recommended to store plasma at -20˚C for short term storage (weeks) or -80˚C for long term storage (months to years) in aliquots to avoid freeze-thaw cycles and to extract cfDNA from plasma only when you are ready to start your NGS library preparation or cfDNA test.
Cell free DNA extraction, storage and quality control (QC) measurements
There are many commercially available kits, both bead and column based, for the extraction of cfDNA from plasma. Page et al (2013) made comparisons between 4 different cfDNA extraction methods and detected wide variation between these in terms of yield. High cfDNA yield does not necessarily mean a better extraction and could indicate the presence of gDNA or carrier RNA which is used in some commercial extraction kits to enhance the recovery of DNA. Appropriate quality controls (QC) are advised and running an aliquot of the extracted nucleic acid on a TapeStation ScreenTape will indicate the level of gDNA/carrier RNA contamination in the sample. Accurate quantification of cfDNA is important for downstream applications including NGS library preparation.
Extracted cfDNA can be stored at -20˚C however freeze-thaw cycles should be avoided, and samples should be accurately quantified once thawed prior to starting your NGS library preparation or cfDNA test.
A successful cell-free DNA assay
Working with cfDNA for either oncology or prenatal applications requires the extremely sensitive and accurate measurement of genomic alterations. The two critical aspects contributing to the success of a genetic test using cfDNA are the quality and quantity of the extracted material as the presence of any gDNA could mask true results. Owing to the relative scarcity of cfDNA, the importance of the pre-analytical steps of sample collection, storage and processing cannot be underestimated (Lippi et al 2011).
More information on pre-analytical products from Nonacus; Cell3™ Preserver, Bead Xtract cfDNA and Cell3™ Xtract is available here:
- Lippi et al. Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med. 2011; 49(7):1113–1126.
- Sato et al. Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy. Oncotarget. 2018; 9(61):31904-31914.
- Lampignano et al. Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows. Clin Chem. 2020; 66(1):149-160
- Page et al. Influence of Plasma Processing on Recovery and Analysis of Circulating Nucleic Acids. PLoS ONE. 2013; 8(10): e77963.