cfDNA Extraction and Quality Control Protocol

Circulating cell-free DNA (cfDNA) is fragmented extracellular DNA, which is released from apoptotic and necrotic cells in small fragments of <200 bp. Cell-free DNA is typically isolated from the bloodstream; however, it is also possible to detect cfDNA in other biological fluids such as urine or cerebrospinal fluid. It has been found that ctDNA of cancer patients carries genetic information from tumor cells. Thus, studying circulating DNA helps to resolve genomic mutations in specific diseases, such as various cancers. It can be used in the future for non-invasive biopsy and monitoring of minimal residual disease (MRD).

Sample Preparation

1. Centrifuge blood collection tubes at 2000 × g for 10 min. If using EDTA tubes, processing time should not be longer than 4 h after sample taking.
2. Carefully move the supernatants (plasma) into a 5 ml tube with a conic bottom without damaging the buffy coat phase.
3. Centrifuge the isolated plasma at 16,000 × g, 4°C for 10 min.
4. Move supernatants to a clean 5 ml tube. Proceed immediately to cfDNA extraction or store at -80°C.
5. cfDNA should be extracted from 4 ml purified blood plasma (set samples to 4 ml by adding phosphate-buffered saline if the volume is less than 4 ml).

cfDNA Extraction

1. Prior to starting the extraction the following should be done:
(a) Equilibrate samples and buffers to room temperature (18–25°C).
(b) Heat a water bath or heating block to 60°C for use with 50 ml tubes.
(c) Heat a heating block to 56°C for use with 2 ml Eppendorf tubes.
2. Pipet 400 μl Proteinase K into a pre-labeled 50 ml tube.
3. Add 4 ml plasma to the tube.
4. Add 3.2 ml buffer ACL to the tube. Mix well by vortexing for 30 s.
5. Incubate for 30 min at 60°C.
6. Add 7.2 ml buffer ACB, and mix well by vortexing for 15–30 s.
7. Incubate the mixture for 5 min on ice.
8. Insert a 20 ml tube extender into the open column. Make sure that the tube extender is firmly inserted into the column to avoid leakage of the sample.
9. Carefully pour the mixture from step 6 into the tube extender. Set the vacuum pump to produce a vacuum of -800 mbar to -900 mbar until all lysates are drawn through (takes up to 15 min).
10. Release the pressure to 0 mbar, discard the tube extenders carefully and leave the columns attached to the VacConnector on the QIAvac 24 Plus.
11. Add 600 μl washing buffer ACW1 to the column. Switch on the vacuum pump (-800 mbar to -900 mbar) while the lid is open. After the entire washing buffer has been drawn through the column, switch the vacuum pump off and release the pressure to 0 mbar.
12. Add 750 μl washing buffer ACW2 to the column. Switch on the vacuum pump (-800 mbar to -900 mbar) while the lid is open. After the entire washing buffer has been drawn through the column, switch the vacuum pump off and release the pressure to 0 mbar.
13. Add 750 μl ethanol (96–100%) to the column. Switch on the vacuum pump (-800 mbar to -900 mbar) while the lid is open. After the entire washing buffer has been drawn through the column, switch the vacuum pump off and release the pressure to 0 mbar.
14. Close the lid of the column. Remove it from the vacuum manifold and discard the VacConnector. Move the column to a clean 2 ml collection tube and centrifuge at full speed (16,000 × g) for 3 min.
15. Move the QIAamp Mini column to a new 2 ml collection tube, open the lid, and incubate at 56°C for 10 min to dry the membrane completely.
16. Move the QIAamp Mini column to a clean 1.5 ml elution tube. Carefully pipet 20–150 μl of Buffer AVE onto the filter of the QIAamp Mini column. Close the lid and incubate at room temperature for 3 min.
17. Centrifuge for 1 min at 16,000 × g. Discard columns.
18. For downstream processing, isolated cfDNA can be stored at 4°C for up to 24 h. For longer storage freeze at <30°C.
19. Use a fragment analyzer to accurately size and qualify the extracted cfDNA.
20. After extraction, cfDNA should be stored at -80 °C.

cfDNA Quality Control by Digital Droplet PCR (ddPCR)

1. Prepare the reaction mixture by adding 10 μl 2× ddPCR Supermix, 0.3 μl forward primer (20 μM), 0.3 μl reverse primer (20 μM), and 0.1 μl probe (20 μM) to 1 μl DNA and fill with ddH2O up to 20 μl.
2. Generate the droplets by pipetting 20 μl sample into the sample wells and 70 μl QX200 Droplet generation oil into the Oil well in the DG8 Cartridge, cover using the DG8 Gaskets and place into the QX200 Droplet generator.
3. Carefully transfer 40 μl of the generated droplets to the Twin. tec PCR Plate by slowly pipetting using a multichannel pipette. Seal plate using a pierceable Foil Heat Seal and a PX1™ PCR Plate sealer.
4. Perform the PCR Reaction on a thermal cycler by incubating the sample for 10 min at 95°C for denaturation followed by 40 cycles of 30 s incubation at 94°C for initial denaturation and 1 min incubation at 60°C for annealing. Apply a final extension step by incubating for 10 min at 98°C and store the sample at 4°C.
5. Move the plates to the QX200 Droplet Reader to measure the amplified Droplets.
6. To measure the amplified fragments, start a new experiment in the QuantaLife™ Software, adjust Supermix to ddPCR™ Supermix w/o dUTP for the desired wells, name targets and select the appropriate channels as used in the probe (FAM, HEX, etc.).
7. Place the plate in the reader and run the experiment, choose DyeSet: FAM or FAM/HEX.
8. Save the analysis and load the exported file into the Quanta- Soft™ Pro software.
9. Set the threshold in the 1D-Amplitude to the positive fraction.
10. Export the results to Excel or any other spreadsheet application.
11. Calculate the mean of the copies/20 μl reads and divide it by 2 to determine the copy number/cell.
12. Divide the copy number/cell by 150 to calculate the concentration (ng/μl). Calculate the cfDNA concentration per 1 ml of plasma.
13. For library preparation, keep the reagents on ice until use.

References:

1. Pott C, Kotrova M, Darzentas N, et al. cfDNA-Based NGS IG Analysis in Lymphoma[M]//Immunogenetics. Humana, New York, NY, 2022: 101-117.
2. Bohers E, Viailly P J, Jardin F. cfDNA Sequencing: Technological approaches and bioinformatic issues. Pharmaceuticals, 2021, 14(6): 596.