Isolation of Circulating Cell-free DNA (cfDNA): A Silica-based Membrane Procedure to Extract cfDNA from Blood Plasma of Cancer Patients

Circulating cell-free DNAs or cfDNAs are short DNA fragments mainly released by cancer cells undergoing apoptosis. cfDNAs are promising cancer biomarkers and can be isolated from body fluids such as plasma.

To isolate cfDNA, first, obtain plasma sample from a cancer patient. Next, treat the plasma with a lysis buffer containing proteinase K, chaotropic salts, and carrier RNA. The chaotropic salts denature proteins, releasing bound cfDNAs from proteins. Proteinase K inactivates nucleases, thereby minimizing cfDNA degradation. 

Thereafter, add the desired binding buffer to the sample. Assemble a spin column containing solid phase silica matrix on the vacuum apparatus. Load the sample in the column. Apply vacuum to pass the sample through the silica membrane.

The carrier RNA non-specifically binds to the tube walls. This non-specific binding along with the presence of salts in the buffer facilitates the binding of cfDNA to the silica membrane, increasing the recovery of cfDNAs. The proteins and other contaminants do not retain on the membrane.     

Now, place the spin column on a collection tube. Centrifuge to remove any residual lysate. Add a suitable washing buffer and centrifuge to remove the salts and any remaining contaminants from the column. Finally, transfer the column to a fresh collection tube and add a suitable elution buffer. Centrifuge to elute the nucleic acids including cfDNAs into the tube.