Abstract
Özet
Amaç. Tek hücre jel elektroforezi veya yaygın olarak kullanılan diğer adı ile Comet Assay tekniği DNA hasarını belirlemek için kullanılan hassas, hızlı ve ucuz bir sitogenetik yöntemdir. Temel olarak bu metod hücrelerin mikroskop lamı üzerinde agaroz içine gömülüp, lizizi sonrası elektroforeze tabi tutularak floresan boyalar ile boyanması ardından DNA’daki hasarın görüntülenmesine dayanmaktadır. Görüntüleme aşamasında interkalatör boyalar olan etidyum bromür ve propidyum iyodit yaygın olarak kullanılmaktadır. Ancak bu boyalar mutajenik ve olası karsinojenik özelliklerinden dolayı kullanım aşamasında oldukça dikkat gerektirmektedir. Bu çalışmada DNA’nın tek hücre jel elektroforezinde görüntülenebilmesi için alternatif olabilecek iki karsinojenik olmayan güvenli boyanın kullanılabilirliği araştırılmıştır. Sağlıklı ve gönüllü bir bireyden alınan periferal kan örneğinden ayrıştırılan lenfositler hücre kültüründe çoğaltılmış, kültür örneği ikiye ayrılarak biri kontrol olarak kullanılmış, diğeri 0,527 kGy/h doz hızlı Cs137 kaynağında 2 Gy ışınlanmıştır. Kontrol ve ışınlanmış örneklere alkalin comet assay yöntemi uygulandıktan sonra lamlar hem etidyum bromür ve propidyum iyodit ile hem de alternatif olarak SafeView ve GelStar boyaları ile boyanmıştır. Bulgular. Elde edilen veriler, klasik boyalarla karşılaştırıldığında bu iki alternatif boyanın son derece etkin çalıştığını göstermiştir. Sonuç. Bu çalışma sonucunda klasik agaroz jel elektroforezi yönteminde DNA ve RNA’yı görüntülemek amaçlı kullanılan SafeView ve GelStar boyalarının, literatürde ilk kez tek hücre jel elektroforezi yönteminde DNA’yı görüntülemek için hassas ve etkin şekilde kullanılabildiği gösterilmiştir.
Anahtar sözcükler: Comet assay, DNA hasarı, floresan boyalar
Abstract
Aim. Single cell gel electrophoresis or the other common name Comet assay technique is a sensitive, fast and cheap cytogenetic method for DNA damage assessment. Method consists of embedding cells to the agarose on microscope slides, electrophoresis following lysis and staining with fluorescent dyes for DNA damage visualization basically. At the staining stage intercalator dyes ethidium bromide and propidium iodide are used widely. However because of the mutagenic and possible carcinogenic properties of these dyes, they must be used very carefully. Method. In this study applicability of two safe non-carcinogenic dyes for DNA visualization in single cell gel electrophoresis is investigated. Lymphocytes from blood sample of a healthy volunteer donor were separated and propagated in cell culture, divided into two, one of them used as control and the other is irradiated 2Gy at a 0,527 kGy/h dose rated 137Cs source. Alkaline comet assay was applied and slides were stained with ethidium bromide and propidium iodide also with alternative SafeView and GelStar dyes. Results. Optained data shows that, when compared with the conventional ones these two alternative dyes worked efficiently. Conclusion. Results of this study showed that SafeView and GelStar dyes which are used in classical agarose gel electrophoresis method for DNA and RNA staining, can also be used efficiently in single cell gel electrophoresis for the first time in literature.
Keywords: Comet assay, DNA damage, fluorescent dyesKeywords
References
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- Verde PE, Geracitano LA, Amado LL, Rosa CE, Bianchini A, Monserrat JM. Application of public-domain statistical analysis software for evaluation and comparison of comet assay data. Mutat Res 2006; 604: 71-82.
- Rydberg B, Johanson KJ. Estimation of DNA strand breaks in single mammalian cells. DNA Repair Mechanism (Hanwalt PC, and Friedberg, EC. Eds), Academic Press, New York 1978; 465-8.
- Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 1984; 123: 291-8.
- Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184-91.
- Olive PL, Banáth JP, Durand RE. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiation Research 1990; 122: 86-94.
- Rojas E, Lopez MC, Valverde M. Single cell gel electrophoresis assay: methodology and applications. J Chromatogr B Biomed Sci Appl 1999; 722: 225- 54.
- Başaran A. Farmakognozide Tek Hücre Jel Elektroforezi Uygulamaları. 14. Bitkisel İlaç Hammaddeleri Toplantısı, Bildiriler, 29-31 Mayıs 2002, Eskişehir, 2002
- Singh NP, Danner DB, Tice RR, Brant L, Schneider EL. DNA damage and repair with age in individual human lymphocytes. Mutation Research 1990; 237: 123- 30.
- Gedik CM, Ewen SW, Collins AR. Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells. Int J Radiat Biol 1992; 62: 313-20.
- Ralph S, Petras M. Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay. Environmental and Molecular Mutagenesis 1997; 29: 418-30.
- Salagovic J, Gilles J, Verschaeve L, Kalina I. The comet assay for the detection of genotoxic damage in the earthworms: a promising tool for assessing the biological hazards of polluted sites. Folia Biol (Praha) 1996; 42: 17-21.
- Verschaeve L, Gilles J. Single cell gel electrophoresis assay in the earthworm for the detection of genotoxic compounds in soils. Bull Environ Contam Toxicol. 1995; 54: 112-9.
- Somorovská M, Szabová E, Vodicka P, Tulinská J, Barancoková M, Fábry R, Lísková A, Riegerová Z, Petrovská H, Kubová J, Rausová K, Dusinská M, Collins A. Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the comet assay with cytogenetic methods and immunology. Mutat Res 1999; 445: 181-92.
- Calderón-Garcidueñas L, Osnaya N, Rodríguez-Alcaraz A, Villarreal-Calderón A. DNA damage in nasal respiratory epithelium from children exposed to urban pollution. Environ Mol Mutagen 1997; 30: 11-20.
- Turner PC, McLennan AG, Bates AD. Instant notes in Molecular Biology. 3 rd ed. New York: Taylor & Francis, 2005; pp: 49.
- Suzuki T, Fujikura K, Higashiyama T, Takata K. DNA staining for fluorescence and laser confocal microscopy. J Histochem Cytochem 1997; 45: 49-53.
Abstract
References
- Olive PL. Impact of the comet assay in radiobiology. Mutat Res 2009; 681: 13- 23.
- Verde PE, Geracitano LA, Amado LL, Rosa CE, Bianchini A, Monserrat JM. Application of public-domain statistical analysis software for evaluation and comparison of comet assay data. Mutat Res 2006; 604: 71-82.
- Rydberg B, Johanson KJ. Estimation of DNA strand breaks in single mammalian cells. DNA Repair Mechanism (Hanwalt PC, and Friedberg, EC. Eds), Academic Press, New York 1978; 465-8.
- Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 1984; 123: 291-8.
- Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184-91.
- Olive PL, Banáth JP, Durand RE. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiation Research 1990; 122: 86-94.
- Rojas E, Lopez MC, Valverde M. Single cell gel electrophoresis assay: methodology and applications. J Chromatogr B Biomed Sci Appl 1999; 722: 225- 54.
- Başaran A. Farmakognozide Tek Hücre Jel Elektroforezi Uygulamaları. 14. Bitkisel İlaç Hammaddeleri Toplantısı, Bildiriler, 29-31 Mayıs 2002, Eskişehir, 2002
- Singh NP, Danner DB, Tice RR, Brant L, Schneider EL. DNA damage and repair with age in individual human lymphocytes. Mutation Research 1990; 237: 123- 30.
- Gedik CM, Ewen SW, Collins AR. Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells. Int J Radiat Biol 1992; 62: 313-20.
- Ralph S, Petras M. Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay. Environmental and Molecular Mutagenesis 1997; 29: 418-30.
- Salagovic J, Gilles J, Verschaeve L, Kalina I. The comet assay for the detection of genotoxic damage in the earthworms: a promising tool for assessing the biological hazards of polluted sites. Folia Biol (Praha) 1996; 42: 17-21.
- Verschaeve L, Gilles J. Single cell gel electrophoresis assay in the earthworm for the detection of genotoxic compounds in soils. Bull Environ Contam Toxicol. 1995; 54: 112-9.
- Somorovská M, Szabová E, Vodicka P, Tulinská J, Barancoková M, Fábry R, Lísková A, Riegerová Z, Petrovská H, Kubová J, Rausová K, Dusinská M, Collins A. Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the comet assay with cytogenetic methods and immunology. Mutat Res 1999; 445: 181-92.
- Calderón-Garcidueñas L, Osnaya N, Rodríguez-Alcaraz A, Villarreal-Calderón A. DNA damage in nasal respiratory epithelium from children exposed to urban pollution. Environ Mol Mutagen 1997; 30: 11-20.
- Turner PC, McLennan AG, Bates AD. Instant notes in Molecular Biology. 3 rd ed. New York: Taylor & Francis, 2005; pp: 49.
- Suzuki T, Fujikura K, Higashiyama T, Takata K. DNA staining for fluorescence and laser confocal microscopy. J Histochem Cytochem 1997; 45: 49-53.
Details
| Primary Language | Turkish |
|---|---|
| Journal Section | Basic Science Research Articles |
| Authors | |
| Publication Date | July 19, 2012 |
| Published in Issue | Year 2012Volume: 34 Issue: 4 |