Where do circulating nucleic acids (cell-free DNA) derive from?
Cells in the human body occasionally undergo apoptosis. During this process, DNA is fragmented and secreted from the cells. Healthy cells, fetal cells, tumor cells, and transplanted cells can all release DNA into the bloodstream. However, the amount of cell-free DNA (cfDNA) in the blood plasma is very low, usually below 10 ng/ml of plasma. Circulating miRNA in blood plasma is either present in exosomes that are secreted by the cells or is associated with RNA-binding proteins. In both cases, the miRNA is prevented from degrading.
Why analyze cell-free DNA?
The analysis of cell-free nucleic acids allows for non-invasive monitoring of disease, the detection of aneuploidy in an unborn child, or the rejection of a transplant. Cell-free nucleic acids are especially promising targets in the field of cancer diagnostics for monitoring disease progression, therapeutic effects of a treatment, or the recurrence of cancer. In addition, analysis of cell-free nucleic acids usually allows for an earlier diagnosis compared to invasive methods, such as common tissue biopsies. Therefore, the analysis of circulating nucleic acids from body fluids is called a "liquid biopsy".
How is cfDNA analyzed?
EDTA blood-draw tubes or Cell-Free DNA BCT (Streck, Inc.) are usually utilized in blood sampling for a "liquid biopsy." After pelleting the cellular components of the collected blood, the supernatant is used for nucleic-acid isolation. Next, quantification of cfDNA is ideally carried out by qPCR or capillary electrophoresis since common methods such as absorption measurement or fluorescent-dye-based quantification might lead to false results due to low DNA concentration. The cell-free nucleic acids are then analyzed by sensitive methods such as quantitative PCR or NGS technologies to detect targets such as miRNA, single nucleotide variants, or chromosome mutations. The new cfDNA products allow for processing of large plasma volumes, which is often required in order to increase the sensitivity of these downstream assays.