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

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Purification of Circulating cfDNA from 1 mL of Plasma

NOTE: This step is performed with a commercial kit (see Table of Materials). All buffers are provided with the kit.

1. Preparation of buffers and reagents
   CAUTION: Do not add acidic solutions or bleach directly to the sample preparation waste. Guanidine salts present in Lysis buffer, Binding buffer, and Wash Buffer-1 when combined with bleach or acids can produce highly reactive compounds.
   1. Binding buffer: Mix 300 mL of Binding buffer concentrate with 200 mL of 100% isopropanol to make 500 mL of working Binding buffer. Store at room temperature.
      NOTE: Binding buffer allows the optimal binding of the circulating nucleic acids to the silica membrane. 500 mL of the binding buffer is sufficient for processing 276, 138, 92, 69 or 55 samples of 1, 2, 3, 4 or 5 mL of plasma respectively and is stable for 1 year at room temperature.
   2. Wash Buffer-1: Mix 19 mL of Wash Buffer-1 concentrate with 25 mL of 96–100% ethanol to make 44 mL of working Wash Buffer-1. Store at room temperature.
      NOTE: Wash Buffer-1 eliminates the contaminants bound to the silica membrane. 44 mL of working Wash Buffer-1 is sufficient for processing 73 samples of 1/2/3/4/5 mL of plasma and is stable for 1 year at room temperature.
   3. Wash Buffer-2: Mix well 13 mL Wash Buffer-2 concentrate with 30 mL of 96–100% ethanol to make 43 mL of working Wash Buffer-2. Store at room temperature.
      NOTE: Wash Buffer-2 eliminates the contaminants bound to the silica membrane. 43 mL of working Wash Buffer-2 is sufficient for processing ~56 samples of 1/2/3/4/5 mL of plasma and is stable for 1 year at room temperature.
   4. To a tube containing 310 μg lyophilized carrier RNA, add 1,550 μL of Elution buffer, to prepare a carrier RNA solution of 0.2 μg/μL. After thoroughly dissolving the carrier RNA, divide the solution to suitable aliquots, and store at -30 °C to -15 °C. Do not freeze-thaw these aliquots more than 3 times. To the Lysis buffer, as shown in Table 1, add the reconstituted carrier RNA dissolved in the Elution buffer.
      NOTE: Because carrier RNA does not dissolve directly in Lysis buffer, it needs to be dissolved first in an Elution buffer and then in Lysis buffer. Firstly, silica membrane-nucleic acids binding is enhanced when there are very few target molecules present in the sample. Secondly, the risk of RNA degradation is reduced because of the presence of large amounts of carrier RNA.
2. Before starting the isolation, bring the columns and samples to room temperature and adjust the sample volumes to 1 mL with sterile phosphate-buffered saline (PBS), if needed. Preheat 2 water baths or heating blocks that contain 50 mL centrifuge tubes and 2 mL collection tubes to 60 °C and 56 °C, respectively.
3. To a 50 mL centrifuge tube, add 100 μL of Proteinase K, 1 mL plasma, and 0.8 mL of Lysis buffer containing 1.0 μg of carrier RNA (prepared in step 1.1.4). Close the centrifuge tube with a cap and mix the contents by pulse-vortexing for 30 s, while ensuring a visible vortex in the tube. Thorough mixing of the contents is important for efficient lysis.
   NOTE: Immediately after vortexing, proceed to step 1.4, without delay.
4. Incubate the solution at 60 °C for 30 min.
5. Remove the cap, add 1.8 mL of the binding buffer to the tube, and thoroughly mix with pulse vortexing for 15–30 s after placing the cap.
6. Incubate the resulting mixture for 5 min on ice and insert the silica membrane column into the vacuum apparatus that is connected to the vacuum pump. Then, firmly insert a 20 mL tube extender into the open column to prevent sample leakage.
7. Carefully pour the incubated mixture into the tube extender of the column and switch on the vacuum pump. After all the lysate mixture completely runs through the columns, switch off the vacuum pump, release the pressure to 0 mbar, and remove and discard the tube extender.
   NOTE: Avoiding cross-contamination, the tube extender should be discarded carefully, to prevent its spreading over adjacent columns.
8. Remove the column from the vacuum apparatus, insert into the collection tube, and centrifuge at 11,000 x g for 30 s at room temperature, to remove any residual lysate. Discard the flow-through.
9. Add 600 μL of Wash Buffer-1 into the column, centrifuge at 11,000 x g for 1 min at room temperature, discard the flow-through.
10. Add 750 μL of Wash Buffer-2 to the column, centrifuge at 11,000 x g for 1 min at room temperature, and discard the flow-through.
11. Add 750 μL of ethanol (96–100%) to the column, centrifuge at 11,000 x g for 1 min at room temperature and discard the flow-through.
12. Centrifuge the column at 20,000 x g for 3 min, by placing it in a clean 2 mL collection tube.
13. Dry the membrane column assembly completely by placing it into a new 2 mL collection tube with the lid open and incubating at 56 °C for 10 min.
14. Place the column in a clean 1.5 mL elution tube. To the center of the column membrane, apply 20–150 μL of Elution buffer and incubate at room temperature for 3 min with the lid closed.
    NOTE: Ensure that the Elution buffer is equilibrated to room temperature. In case of using elution buffer less than 50 μL, ensure that it is dispensed carefully onto the center of the membrane. This helps with the complete elution of the bound DNA. However, the elution volume is not fixed and can be changed as per the downstream applications. The recovered eluate can be up to 5 μL and certainly less than the elution volume applied to the column.
15. Centrifuge the recovered eluate in a microcentrifuge at 20,000 x g for 1 min to elute the nucleic acids, and store at -20 °C.

Table 1. Volumes of Lysis buffer and carrier RNA (dissolved in Elution buffer) required for processing 1 mL plasma samples.