Handling cfDNA
cfDNA isolation methods and protocols
You can use either silica membrane-based spin columns or magnetic bead technology for cfDNA purification. Regardless of the cfDNA isolation method, we recommend using a cfDNA purification kit with a lysis step that releases nucleic acids that are bound to proteins and lipids and contained in vesicles (1).
Some cfDNA purification protocols include the option of adding carrier RNA before extraction to increase the yield of cfDNA, but carrier RNA may also negatively affect downstream analysis (2).
The input volume for cfDNA isolation protocols can vary from 1 to 10 mL, depending on the target DNA, the application, and the downstream assay‘s sensitivity. Starting volumes of 4 mL are usually used, which is the total volume of plasma from one blood collection tube.
QIAsymphony uses magnetic bead technology to automated cfDNA isolation. Recommended purification kits
Our cfDNA purification kits provide fast workflows and isolate high-quality cfDNA for high sensitivity in downstream NGS and PCR analyses. The QIAamp Circulating Nucleic Acid Kit has come to be recognized by the scientific community as the gold standard for cfDNA isolation, while the QIAamp MinElute ccfDNA Midi Kit is the second generation product that allows processing of up to 10 mL serum or plasma and can be partially automated on QIAcube Connect.
Automated cfDNA purification
Automated cfDNA purification methods reduce variability and increase throughput while reducing hands-on time. In contrast to basic research applications, automated cfDNA purification is often the preferred choice in translational research and in the diagnostic routine, as it ensures a standardized workflow with high traceability and LIMS compatibility, operator-independent reliability and optimal use of time and resources.
A fully automated workflow also offers the advantages of using a dedicated cfDNA purification kit, including:
- Optimized purification chemistry
- Prefilled reagent cartridges with bar codes for safety and ease of use
- Reduced risk of exposure and sample mix-up
- Highest reproducibility
Determining background
When profiling very low-abundance targets such as biofluid cfDNA, it is important to ensure the signals you are measuring are reliably above any background signal. A blank purification, for example, with water added in place of a biofluid sample in the cfDNA purification, can be used as a negative control to measure any background signal.
cfDNA storage
Store purified cfDNA at –15 to –30°C or –65 to –90°C in RNase-free water or manufacturer-supplied elution buffer. Under these conditions, no degradation of DNA is detectable after one year and cfDNA stability is maintained.
2. Gyanchandani, R. et al. (2018) Whole genome amplification of cell-free DNA enables detection of circulating tumor DNA mutations from fingerstick capillary blood. Sci. Rep. 8 (1).