Introduction to cell-free DNA

Challenges in cfDNA analysis

What are the main cfDNA challenges? Molecular analysis of cfDNA in biofluid samples is complicated by various challenges. First, the analyte is only available in very low and highly variable concentrations, ranging from 1–50 ng/mL in healthy individuals.  

cfDNA is also highly fragmented and often degraded, affecting downstream molecular analyses. Furthermore, the target cfDNA molecules for analysis are typically diluted by a higher-abundance background of wild-type or maternal cfDNA and genomic DNA, which makes it difficult to detect the marker of interest. 

The potential for experimental bias through contamination with cellular DNA or substances that inhibit downstream analyses is high, so it is crucial to minimize this risk.  

During the storage and transport of blood samples, cells can lyse and release DNA, making it appear that the sample has a higher level of cfDNA than it actually does. The type of anticoagulant and crosslinking reagents in blood collection tubes can impact the quality of cfDNA and the end results (4).

Cell-free DNA troubleshooting


Five key pitfalls to avoid in cfDNA isolation

  • Lack of stabilization during sample collection 

  • Inadequate storage or transportation concept 

  • Suboptimal centrifugation parameters 

  • Cumbersome or inappropriate cfDNA purification method 

  • Suboptimal methods for quality or yield quantification 

1. Moss, J. et al. (2018) Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nat. Commun. 9 (1). 
2. Bronkhorst, A.J., Ungerer, V., and Holdenrieder, S. (2019) The emerging role of cell-free DNA as a molecular marker for cancer management. Biomol. Detect. Quantif. 17. 
3. Stewart, C.M., and Tsui, D.W.Y. (2018) Circulating cell-free DNA for non-invasive cancer management. Cancer Genet. 228–229: 169–179. 
4. Voss, T., Ullius, A., Schönborn, M., and Oelmüller, U. (2021) Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal. PLoS ONE 16 (7).
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