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.

To isolate circulating cell-free DNA from a patient plasma sample, add 100 microliters of Proteinase K and 800 microliters of Lysis buffer supplemented with one microgram of carrier RNA to 1 milliliter of patient plasma. Thoroughly pulse vortex the solution for 30 seconds before incubating for 30 minutes at 60 degrees Celsius. At the end of the incubation, add 1.8 milliliters of binding buffer to the tube and thoroughly mix with 15 to 30 seconds of pulse vortexing.

After a 5-minute incubation on ice, insert a silica membrane column into a vacuum apparatus connected to a vacuum pump and firmly insert a 20-milliliter tube extender into the open column to prevent sample leakage. At the end of the incubation, carefully pour the mixture into the tube extender and switch on the vacuum pump. When all of the lysate has completely run through the columns, switch off the vacuum pump, release the pressure to zero millibars, and carefully discard the tube extender without contaminating the adjacent columns.

Transfer the column into a collection tube for centrifugation to remove any residual lysate. After discarding the flow-through, add 600 microliters of Wash Buffer-1 to the column for a second centrifugation. After discarding the flow-through, centrifuge the column again with 750 microliters of Wash Buffer-2. After discarding the flow-through, add 750 microliters of 96% to 100% ethanol to the column for an additional centrifugation.

Transfer the column into a new 2-milliliter collection tube for another centrifugation before placing the membrane column assembly into a new 2-milliliter collection tube at 56 degrees Celsius for 10 minutes. At the end of the incubation, transfer the column into a new 1.5-milliliter elution tube and add 50 microliters of elution buffer to the column for a 3-minute incubation at room temperature. Then, centrifuge the recovered solution for 1 minute at 20,000 x g to elute the nucleic acids.