Isolation of micronuclei: high yield by sucrose gradient versus maximum purity by cell sorting
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Published on: September 20, 2021
M Abend1, S Frombeck, C Baaske
1Institute of Radiobiology, Federal Armed Forces Medical Academy, Munich, Germany 100305.3537@compuserve.com
Abstract:
Micronuclei (MNC) of L929 cells were isolated 72 h after irradiation with 6 Gy for characterization of their DNA content, using gel electrophoresis. A novel method for isolation of MNC based on a sucrose gradient ultracentrifugation was developed. With this efficient method (80% recovery) more than 150 x 10(6) MNC per day could be isolated. The purity was > 99%. However, the low number of main nuclei in the MNC isolate ( < 1%) resulted in a contamination of MNC DNA with about 15% main nucleus DNA, due to the several times higher DNA content of main nuclei. Cell sorting was utilized to maximize the purity by choosing the recommended sorting mode for highest purity. Isolation of MNC with the cell sorter was successful (100% purity), but also time-consuming (1-2 x 10(6) MNC per working day could be isolated) and insufficient (10% recovery). Extraction of DNA of these isolated MNC resulted in less than 1 ng/day. Hence, at least 1 week of cell sorting would be necessary for one electrophoretic run. When employing the sucrose gradient method, 2000 times more DNA of MNC have been isolated. We therefore consider this method as the most efficient way for rapid and low cost isolation of large amounts of purified ( > 99%) MNC without the employment of sophisticated and expensive techniques (cell sorting) and the accompanied knowhow. In contrast, maximum purity but low yields of MNC can be obtained by cell sorting.
Insights
A novel sucrose gradient method efficiently isolates micronuclei (MNC) for DNA analysis, yielding high purity and quantity. This cost-effective technique surpasses cell sorting for large-scale MNC DNA isolation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Micronuclei (MNC) are critical biomarkers for genotoxicity and chromosomal instability.
- Accurate DNA content characterization of MNC requires highly purified isolates.
- Existing isolation methods often face limitations in efficiency, purity, or cost.
Purpose of the Study:
- To develop and evaluate a novel, efficient method for isolating micronuclei (MNC) from L929 cells.
- To compare the developed method with cell sorting for purity, yield, and cost-effectiveness.
- To facilitate large-scale DNA isolation from MNC for downstream applications like gel electrophoresis.
Main Methods:
- Development of a novel isolation technique for MNC using sucrose gradient ultracentrifugation.
- Characterization of isolated MNC DNA content 72 hours post-irradiation (6 Gy) via gel electrophoresis.
- Comparative analysis with cell sorting for MNC isolation efficiency and purity.
Main Results:
- The sucrose gradient method achieved >99% purity and 80% recovery, isolating over 150 x 10^6 MNC daily.
- This method yielded 2000 times more MNC DNA compared to cell sorting.
- Cell sorting provided 100% purity but was time-consuming with low recovery (10%) and minimal DNA yield (<1 ng/day).
Conclusions:
- Sucrose gradient ultracentrifugation is a highly efficient, rapid, and cost-effective method for isolating large quantities of purified MNC.
- This technique avoids the need for sophisticated and expensive equipment like cell sorters.
- The developed method is superior to cell sorting for large-scale MNC DNA isolation required for analyses.
