DNA Preparation of Bone Marrow Sample for Backtracking

Backtracking plays an essential role in leukemia research. I have talked about the backtracking method in my previous post. Now, we are going to discuss step by step about backtracking method that used in leukemia research. We start off with the DNA preparation of bone marrow sample first before we go any further.

Preparation of DNA

The preparation of DNA from bone marrow sample for backtracking is not easy to be done. Yet, we need the high molecular weight DNA to do the research on translocations and deletions. There are many things that we need to know before the preparation of DNA starting. For backtracking small rearrangements like IGH rearrangements or point mutations, any size of the DNA is sufficient for cancer research. If we manage to get the fresh bone marrow sample, then the mononuclear cells should be purified in order to clear off the neutrophils for purer blast cell preparation. In fact, we still can get the DNA from frozen bone marrow for the cancer research too. If we use the cryopreserved mononuclear cells, we need to wash the cells once before trying to get the high molecular weight DNA from the cells. However, it is not recommended to use the repeated freeze and thawing of bone marrow sample and DNA for this cancer research.

Isolate Mononuclear Cells

To isolate the mononuclear cells, there are many steps that we need to follow.

For Fresh Bone Marrow

1. Firstly, the bone marrow sample was diluted with 3 X RPMI-1640 and carefully layer on top of a 1/3 volume Ficoll.

2. The tubes were spun at 400X ϑ for 30 minutes with the brake off.

3. Next, the mononuclear cell layer above the Ficoll was removed with a transfer pipette.

4. The cells were added to 10ml PBS and spun at 200X ϑ.

5. The supernatant was discarded.

For Frozen Bone Marrow

1. Firstly, the frozen bone marrow was diluted 1:1 in phosphate buffered saline (PBS) to aid the lysis of red blood cell.

2. The mixture was spun at 3500X ϑ and the red supernatant was poured off. We can see the pellet still consists of sticky lysed cells. If there is no visible pellet, the degraded DNA may be isolated from supernatant.

Getting High Molecular Weight DNA

1. The pelleted fresh cells or frozen cell were vortex at low speed in order to disperse the pellet.

2. 500µl of 1:1 PBS was added to around 2 X 10⁶ cells in the pellet and mix with wide bore pipette.

3. To isolate DNA from supernatant, the 500µl of 2X concentration of PBS is used.

4. 500µl of PCI was added into the tube for 30 minutes.

5. Later, the tube was spun at 10000X ϑ for 10 minutes.

6. The aqueous supernatant was removed to a new tube without disturbing the PCI layer.

7. The PCI layer was discarded and add in 1:10 volume of 10M NH₄OAc into the tube to mix.

8. Next, the 2X volume of ethanol was added and mixed up the ethanol and precipitated DNA.

9. The ethanol was removed from the mixture. The precipitation was washed one time with 500µl 70% ethanol.

10. Again, the ethanol was removed by pipette of Kimwipe.

11. 200µl of TE was added and let DNA dissolve for one day prior use.

12. Lastly, the DNA quantification was done.

Getting Low Molecular Weight DNA and Leukemia Cells

There are some DNA purification kits such as Qiagen Micro and Mini kits that use columns and beads to shear DNA through physical and hydrodynamic forces. Although this kind of method is good in isolating high-quality DNA of any quantity, the molecular weight of the DNA will be lessen. The results produced are good for PCR-based assays to detect IGH and TCR rearrangements and point mutations that present in leukemia oncogenes.

That is all about the DNA preparation of bone marrow sample for leukemia research. It is not easy to do this as the bone marrow cells are too fragile. One of the aims of my research is to culture the bone marrow cells successfully. I think I must do more reading in order to advance my knowledge in this field.

(Reference: Leukemia edited by Chi Wai Eric So)