Circulating Nucleic Acids in Human Biofluids and Liquid Biopsy Research

Several different types of nucleic acids can be found circulating in human biofluids. Fragmented DNA and RNA are now routinely purified from plasma and other bodily fluids. These types of nucleic acids need to be purified from a cell-free fraction of the biofluids to ensure that the isolated nucleic acids are truly circulating and not from intact cells. In this blog post, we will learn a bit more about circulating nucleic acids (CNA) and how they can be used as biomarkers in research.

Circulating Cell-free DNA

Circulating, cell-free DNA (ccfDNA), also called cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), was first described in 1948 (1). ccfDNA is typically associated with histone proteins and fragmented to nucleosome size of about 170bp. The half-life of ccfDNA is short, approximately 30 minutes, and it is constantly being degraded or removed and replenished. Normal concentrations of ccfDNA are approximately 2–20ng/ml of plasma. This equates to roughly 300–3000 genomic equivalents/ml of plasma.

ccfDNA can be used to detect tumor DNA in oncology research, and the fetal fraction of maternal plasma (cell-free fetal DNA, cffDNA) can be used to detect genetic disorders in non-invasive, pre-natal testing (NIPT). The effectiveness of using ccfDNA for these areas of study is well known in liquid biopsy research, and this less invasive approach is preferable because there is no need for a tissue biopsy (surgery) for cancer detection, or amniocentesis for fetal testing.

Circulating Cell-free RNA

There are also several different types of cell-free RNA present in biofluids. Limited messenger RNA (mRNA) or mRNA fusions can be detected within the population of circulating total nucleic acids found in cell-free biofluid samples. Several types of small RNA are emerging in biomarker research including micro RNA (miRNA), piRNA, tRNA fragments (tRFs), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and long noncoding RNA (lncRNA) (2). All of these are typically associated with proteins in their functions. These proteins help protect them from RNase activity, which can be very active in some biofluid sample types.

First described in 1993, miRNAs are relatively small RNA with a size of approximately 17–24nt (3).  They are often associated with argonaute proteins in the RNA-inducing silencing complex (RISC) and are involved in post-transcriptional regulation. miRNA combined with RISC can be found circulating freely or within extracellular vesicles called exosomes. Exosomes are 30–150nm in size and have been shown to allow for cross-talk between cells, sometimes at distant sites from the source of the exosome (4).

Fluids That Can Contain Circulating Cell-Free DNA or RNA

The biofluids that contain CNAs can be very diverse. From blood, typically plasma is the best source of CNA, especially if the plasma is spun in a centrifuge soon after the blood collection.  To limit the risk of potential genomic DNA contamination, it is important to centrifuge the plasma twice to ensure that all white blood cells have been isolated and removed. Serum also contains CNA, however there is typically an apoptotic DNA ladder that results in fragments in the same size range and cell-free, genomic DNA. Urine has become another important biofluid for non-invasive testing. ccfDNA purified from urine typically called transrenal DNA (trDNA). Other biofluids used in CNA research include saliva, milk, tears and gastric juices.

As new purification techniques and more complex downstream assay become available, the field of circulating nucleic acid research will continue to evolve. The promise of these biomarker discovery efforts is that they can lead to more diagnostic information coming from less invasive sample collection methods. Information from CNA could also accelerate the development of therapeutic treatments by allowing researchers to monitor responses using less invasive methods (5).


  1. Mandel P, Metais P. (1948) Les acides nucleiques du plasma sanguin chez l’homme. C R Seances Soc Biol Fil., 142, 241–3.
  2. Dunaeva, M. and Pruijn, G.J.M. (2020). Global Characterization of Circulating Nucleic Acids. In: Astakhova, K., Bukhari, S. (eds) Nucleic Acid Detection and Structural Investigations. Methods in Molecular Biology, vol 2063. Humana, New York, NY.
  3. Lee, RC., et al. (1993) The C. elegans Heterochronic Gene lin-4 Encodes Small RNAs with Antisense Complementarity to lin-14. Cell 75, 843–54.
  4. Park, J.H. and Kornfeld, JW. (2021)  ExomiRs at the crossroads—divergent role of exosomal miRNAs in early and chronic obesity. Nat Metab. 3, 1137–8.
  5. Liquid Biopsies Guide the Development of Cancer Drugs. GEN Genetic Engineering and Biotechnology News, 42 published online September 6, 2022. Accessed September 26, 2022.[DH1] 

Discover More in our ccfDNA webinar series: The Basics and Beyond
Part 1: ccfDNA 101: Emerging Trends in Oncology Research
Part 2: ccfDNA Workflows: Honing in on the Target
Part 3: ccfDNA in the Lab: Optimizing Purification for Sequencing

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Douglas Horejsh
Douglas Horejsh is Associate Director, R&D, at Promega leading the group that develops the nucleic purification chemistries. He received his BS in Biochemistry/Molecular Biology from the University of Wisconsin - Eau Claire and his PhD in Cell and Molecular Biology from the University of Wisconsin - Madison.
Douglas Horejsh

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