DNA preparation from cryopreserved bone marrow cell samples.
1. Quickly thaw cryopreserved MNCs (1 ml) in 9 ml PBS supplemented with DNase I immediately before use (final concentration: 0.1 mg/ml). 2. Incubate at 37 degrees C for 10 min. 3. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 4. Incubate with saturating amounts of selected MoAbs for 20 min at 4 degrees C. 5. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 6. Immunomagnetic bead separation of viable cells as described by Lea et al. (1). 7. DNA extraction. 8. Measure quality and yield of DNA by spectrophotometry. In conclusion, the DNA extraction method presented here, based on DNase I pretreatment of the cryopreserved cells followed by an immunological selection for viable cells, provides cell suspensions with close to 100% viability; thus, high-quality DNA can be extracted even from cryopreserved cell samples of low initial viability. Furthermore, using this method DNA analyses can be performed on selected cellular subsets if desired. We thus recommend this method for DNA analyses in hematological research when the only materials available are cryopreserved bone marrow samples of low viability.
Jørgensen
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Institute of Medical Microbiology, University of Aarhus, Denmark.
Hokland
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Petersen
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Hokland
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D001853:Bone Marrow / Q000737:chemistry* / Q000276:immunology; D001854:Bone Marrow Cells; D016923:Cell Death / Q000276:immunology; D015925:Cryopreservation; D004247:DNA / Q000302:isolation & purification*; D003850:Deoxyribonuclease I / Q000737:chemistry; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D013997:Time Factors
D004247:DNA; D003850:Deoxyribonuclease I