Kichukova, Natasa and Milev, Mishko and Janeva, Milikca and Maksimova, Viktorija and Ruskovska, Tatjana (2019) Optimization of silver staining method in single cell gel electrophoresis (Comet assay). In: Second congress of students of general medicine, 09-10 May 2019, Stip, Republic of North Macedonia.
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Abstract
Introduction
The comet assay is one of the most common methods for measuring genotoxicity in cells. In 1984, Ostling and Johanson demonstrated movement of DNA strands when nuclei were exposed to an electric field1.
Aim
Optimization of silver staining method in single cell gel electrophoresis
Materials and methods
Lysed cells are embedded in low melting agarose on a microscope slide, pre-coated with normal melting agarose. When they are subjected to electrophoretic field, the damaged parts of the DNA travel toward the anode, and when stained and examined microscopically the images resemble comets. The undamaged coiled DNA can be found in the “head” of the comet, whereas the comet “tail” presents the damaged DNA. The DNA damage can be estimated by measuring the head diameter, tail length and tail momentum.
Results
An alternative method to the commonly used florescent one, is the usage of silver staining. The optimization of silver staining method in single cell electrophoresis has shown many critical variables which should be considered when applying this type of staining. High purity of AgNO3 solution, high cleanness of all laboratory glass, time of fixation, time of staining, shaking during the staining, freshness of staining solution and light exposure are very important and critical parameters that are referring to this method.
Conclusion
Having in mind these critical points, silver staining can produce excellent quantification of DNA damage with comet assay using only standard light microscopy.
| Item Type: | Conference or Workshop Item (Poster) |
|---|---|
| Uncontrolled Keywords: | DNA, silver staining, comet assay |
| Subjects: | Medical and Health Sciences > Other medical sciences |
| Divisions: | Faculty of Medical Science |
| Depositing User: | Viktorija Maksimova |
| Date Deposited: | 13 Jun 2019 09:01 |
| Last Modified: | 13 Jun 2019 09:01 |
| URI: | https://eprints.ugd.edu.mk/id/eprint/22101 |
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