Hello Marah Amer,

Yes, contamination of gDNA would introduce serious background noise and lead to inaccurate result.

If you want to avoid gDNA contamination in cfDNA samples, the ideal way would be to perform centrifugation of blood and cfDNA extraction as soon as the blood sample is collected, which would prevent the release of gDNA to a greatest extent. Special care should be taken to avoid transfer of any other blood components (buffy coat or red blood cells), when separating the plasma fraction.

You may also make use of blood collection tubes specialized for cfDNA preservation which are commercially available with stabilizers to minimize the cellular gDNA release, inhibit cfDNA degradation from nuclease and maintain the overall stability of cfDNA. With proper operation in certain condition, these tubes could achieve the preservation of cfDNA for about 7 days.

There is also another way to protect the cfDNA from gDNA contamination. You may prevent the rupture of cells with cross-linking agents such as formaldehyde. I would not recommend this method because formaldehyde can lead to cross-linking between cfDNA and proteins, which brings in other contamination thus lowering the quality of extracted cfDNA and more severely, destroying it.

Generally, cfDNA circulates in fragments ranging between 120–220 bp, with a short half-life (5-150 min), and should be extracted as soon as possible.

Since cfDNA is shorter than gDNA, one can selectively extract DNA below a size threshold to remove gDNA contamination. The use of nano-magnetic particles can provide higher surface area and enhanced kinetics, and are suitable for application in such case since cfDNA is significantly shorter and lower in concentration than gDNA.

You may follow the workflow provided below in four steps.

1. Mix nano-magnetic particles with your sample.

2. When mixed with the sample, the nano-magnetic particles will bind to the targeted cfDNA in the sample.

3. You may use a magnetic force to pull and aggregate the bound material. You may remove the unbound materials by aspiration. What remains is the nanoparticle-bound target namely, cfDNA.

4. Finally, you may release the bound target material (cfDNA) from the nanoparticles.

Based on these four steps, you may design your protocol and get your cfDNA sample free of gDNA contamination.

You may want to refer to the article attached below. It may be helpful!

https://www.sciencedirect.com/science/article/abs/pii/S1570023222001404

Best.

Malcolm Nobre I really appreciate your answer, Thank you!