DNA Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue

DNA Extraction from FFPE Tissue with Genomic DNA Isolation Kit

1. Place two to five 10 μm sections of FFPE tissue (of approximately 1 cm² in size) into a 1.5 ml Eppendorf tube.
2. Add 1000 μl xylene and vortex thoroughly for 10 s (work in a protective cabinet).
3. Centrifuge for 5 min, 20,000 × g.
4. Remove the supernatant carefully and dispense it in specific waste containers.
5. Add 1000 μl 99% ethanol.
6. Centrifuge for 5 min, 20,000 × g.
7. Remove carefully the ethanol.
8. Add again 1000 μl 99% ethanol and mix carefully by inverting the tube.
9. Centrifuge for 5 min, 20,000 × g.
10. Remove all ethanol and air-dry the remaining tissue by leaving the tube open, and incubate for 10–15 min at 37 °C in a thermo block.
11. Resuspend the pellet in 180 μl Buffer ATL.
12. Add 20 μl proteinase K, and mix by vortexing.
13. Incubate overnight at 56 °C.
14. Incubate at 90 °C for 1 h.
15. Add 200 μl Buffer AL to the sample, and mix thoroughly by vortexing.
16. Add 200 μl ethanol (96–100%), and mix again thoroughly by vortexing.
17. Continue with the extraction and DNA purification, before starting the sample preparations for clonality assessment.

DNA Extraction from FFPE Tissue Starting with the Chelex Method

1. deparaffinized0 μm tissue sections until step 10.
2. Add 200 μl of 5% Chelex-100 homogeneously mixed in TET lysis buffer.
3. Incubate for 5 min at 95 °C in a thermo shaker at 350 rpm.
4. Cool down for 5 min at room temperature.
5. Add 20 μl of proteinase K and incubate o/n at 56 °C in a thermo shaker at 350 rpm.
6. Incubate for 10 min at 95 °C in a thermo shaker at 350 rpm.
7. Centrifuge for 10 min, 20,000 × g at room temperature.
8. Transfer the supernatant to a clean 1.5 ml Eppendorf tube.
9. Centrifuge for 10 min, 20,000 × g at room temperature.
10. Transfer the supernatant to a clean 1.5 ml Eppendorf tube.
11. Add 180 μl ATL buffer (QIAamp DNA Micro Kit), vortex for 10 s, and incubate at room temperature for 30 min.
12. Incubate at 80 °C for 10 min, and then allow to cool down to room temperature.
13. Briefly centrifuge the 1.5 ml tube to remove drops from the inside of the lid.
14. Add 200 μl buffer AL and vortex briefly.
15. Incubate at 70 °C for 10 min, and then allow to cool down to room temperature.
16. Add 250 μl of ethanol 96%, vortex briefly.
17. Continue the procedure with the DNA purification, before starting the sample preparations for clonality assessment.

DNA Purification with QIAamp DNA Microcolumn

1. Carefully transfer the entire lysate to the QIAamp MinElute column (in a 2 ml collection tube) and centrifuge at 6000 × g for 1 min.
2. Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the collection tube containing the flow-through.
3. Add 500 μl Buffer AW1 on the column and centrifuge at 6000 × g for 1 min.
4. Discard the flow-through and add 500 μl Buffer AW2 to the column.
5. Centrifuge at 6000 × g for 1 min and discard the flow-through.
6. Centrifuge at full speed (20,000 × g) for 3 min to dry the membrane completely.
7. Place the QIAamp MinElute column in a clean 1.5 ml Eppendorf tube, and discard the collection tube containing the flow-through.
8. Apply 20–100 μl Buffer ATE (QIAamp DNA FFPE Kit) or 20–100 μl Buffer AE (QIAamp DNA Micro Kit) to the center of the column membrane.
9. Incubate at room temperature for 5 min.
10. Centrifuge at full speed (20,000 × g) for 1 min.
11. Discard the column and keep the 1.5 ml tube containing the DNA solution.
12. Determine the DNA concentration and dilute if necessary to a working solution of 20–40 ng/μl with the used elution buffer or MQ.

Reference:

1. van Bladel D A G, van der Last-Kempkes J L M, Scheijen B, et al. Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma[M]//Immunogenetics. Humana, New York, NY, 2022: 7-42.