The method is simple and is commonly used in biomonitoring and evaluating the effects of the various toxins on the DNA. One of the challenges with the method is that it requires fluorescent staining and specialized microscopes capable of detecting fluorescence. It was possible to use the regular bright-field microscopy using a silver staining technique, but this method is still comparatively expensive and complicated to be used in the many resource-constrained laboratories.
A team of Russian and Canadian scientists from the Burnasyan FMBC and the Atomic Energy of Canada modified the DNA comet assay turning it into a cost-effective and method that may be used in some of the poorest geographies while maintaining high quality research standards.
Partly by accident, Dr. Andreyan Osipov, the lead author on the study, faced with a need to perform an experiment and absence of fluorescent reagents tried several staining techniques to adapt DNA comet assay to bright-field microscopy and found that the simple and widely-available Giemsa staining facilitated for unexpectedly high sensitivity.
The international team was formed to develop and test the assay. The experiments carried out by the team demonstrated that
Giemsa stain had similar sensitivity as the fluorescent SybrGreen I, while providing for better photo-stability and higher resolution of the bright-field microscopy.
“I never expected that the regular Giemsa stain, which is a mixture of azure and eosin, broadly used in the many laboratories worldwide could compare to the fluorescent dyes. We tried almost every other dye available to see if we can achieve better results.
I truly hope that our modification of the standard DNA comet assay will help scientists around the world develop new applications for it and make this method more popular”, said Dr. Andreyan Osipov, head of the radiation biophysics laboratory at the Burnasyan Federal Medical Biophysical Center fo Federal Medical Biological Agency in Russia.
In series of experiments scientists took lymphocytes from peripheral blood, irradiated cells with 3 and 6 Gy and analyzed the comets obtained with Giemsa staining usind the CASP software.
The sensitivyty of SybrGreen I was only 1.2 fold more sensitive than Giemsa staining.
“When it comes to evaluating the roadblocks in scientific progress most scientists and activists including myself are pinpointing lack of public funding as the main bottleneck. However, it is the lack of qualified talent, which impedes the many important areas such as biogerontology and regenerative medicine. And to get more qualified specialists we need to mobilize the human capital in developing geographies like Africa and India. The development of cost-effective and readily-accessible methods to measure DNA damage and repair is one step forward towards this ambitious goal. I am very surprised that after three decades of working with the classic DNA comet method no one realized that it can be performed in a high school setting or used in bedside medicine in remote areas using a simple Giemsa staining and the common bright-field microscope”, commented Alex Zhavoronkov, PhD, the director of the Biogerontology Research Foundation in the UK.
The study was published in the International Journal of Molecular Science, and is freely available as an Open Access publication.
DNA Comet Giemsa Staining for Conventional Bright-Field Microscopy
Osipov A, Arkhangelskaya E, Vinokurov A, Smetaninа N, Zhavoronkov A, Klokov D. International Journal of Molecular Sciences. 2014; 15(4):6086-6095.
Open Access: http://www.mdpi.com/