Use of SYBR Safe Gel Stain (Hibrigen) in Comet Assay: Short-Term, Low-Cost Protocol

Yıl 2025, Cilt: 1 Sayı: 3, 82 - 87, 29.11.2025

Mehmet Tahir Hüsunet , İbrahim Halil Kenger , Batuhan Bilgin

https://doi.org/10.65308/gjohbs.2025.012

Öz

In this study, the applicability of SYBR Safe gel stain in the Comet Assay protocol was evaluated, and staining conditions were optimized. The mutagenic and toxic properties of ethidium bromide (EtBr) have increased the need for alternative fluorescent dyes. In the study using human SH-SY5Y neuroblastoma cell line, DNA damage was induced by H₂O₂ and analyzed by fluorescence microscopy after staining with SYBR Safe. Different SYBR Safe concentrations (1:1000-1:10000) and staining times (2-30 min) were tested. The optimum signal to image ratio was obtained with a dilution of 1:2500 and an incubation time of 5 min. Furthermore, 0.1 M NaCl solution was found to be the most suitable carrier medium in terms of dye stability and fluorescence quality. It was observed that the prepared preparations were stable at +4°C for up to 1 month. These results show that SYBR Safe is not only a safe alternative but also provides high image quality and cost advantage with low concentration. The findings demonstrate that SYBR Safe is a safe, economical, and high-performance alternative to EtBr in comet assay applications.

Anahtar Kelimeler

Comet Assay , SYBR Safe Gel Stain (Hibrigen) , DNA Staining , Ethidium Bromide Alternative

Comet Assay’de SYBR Safe jel Boyası (Hibrigen) Kullanımı: Kısa Süreli, Düşük Maliyetli Protokol

Öz

Bu çalışmada, SYBR Safe jel boyasının Comet Assay protokolündeki uygulanabilirliği değerlendirilmiş ve boyama koşulları optimize edilmiştir. Etidyum bromürün (EtBr) mutajenik ve toksik özellikleri, alternatif floresan boyalara olan ihtiyacı artırmıştır. İnsan SH-SY5Y nöroblastoma hücre hattı kullanılarak gerçekleştirilen çalışmada, H₂O₂ ile DNA hasarı oluşturulmuş ve SYBR Safe ile boyama sonrası floresan mikroskopi ile analiz yapılmıştır. Farklı SYBR Safe konsantrasyonları (1:1000–1:10000) ve boyama süreleri (2–30 dk) test edilmiştir. En uygun sinyal/görüntü oranı 1:2500 seyreltim ve 5 dakikalık inkübasyon süresiyle elde edilmiştir. Ayrıca, 0.1 M NaCl çözeltisinin boyanın stabilitesi ve floresan kalitesi açısından en uygun taşıyıcı ortam olduğu belirlenmiştir. Hazırlanan preparatların +4 °C'de 1 aya kadar stabil kalabildiği gözlemlenmiştir. Bu sonuçlar, SYBR Safe’in yalnızca güvenli bir alternatif olmadığını, aynı zamanda düşük konsantrasyonla yüksek görüntü kalitesi ve maliyet avantajı sağladığını göstermektedir. Elde edilen bulgular, Comet Assay uygulamalarında SYBR Safe’in EtBr’ye kıyasla güvenli, ekonomik ve yüksek performanslı bir seçenek olduğunu ortaya koymaktadır.

Anahtar Kelimeler

Comet Assay , SYBR Safe Jel Boyası (Hibrigen) , DNA Boyama , Etidyum Bromür Alternatifi

Kaynakça

• 1. Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp. 2012(62).
• 2. Nass MM. Differential effects of ethidium bromide on mitochondrial and nuclear DNA synthesis in vivo in cultured mammalian cells. Exp Cell Res. 1972;72(1):211-22.
• 3. Haugland RP. Handbook of fluorescent probes and research products: Molecular probes; 2002.
• 4. Cosa G, Focsaneanu KS, McLean JR, McNamee JP, Scaiano JC. Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution. Photochem Photobiol. 2001;73(6):585-99.
• 5. Ohta T, Tokishita S-i, Yamagata H. Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2001;492(1-2):91-7.
• 6. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental cell research. 1988;175(1):184-91.
• 7. Kumaravel TS, Vilhar B, Faux SP, Jha AN. Comet Assay measurements: a perspective. Cell Biol Toxicol. 2009;25(1):53-64.
• 8. Gandhi VP, Kesari KK, Kumar A. The Identification of Ethidium Bromide-Degrading Bacteria from Laboratory Gel Electrophoresis Waste. BioTech (Basel). 2022;11(1).
• 9. Singer VL, Lawlor TE, Yue S. Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test). Mutat Res. 1999;439(1):37-47.
• 10. Shaker GH, Melake NA. Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies. Saudi Pharm J. 2012;20(3):221-7.
• 11. Collins A, Moller P, Gajski G, Vodenkova S, Abdulwahed A, Anderson D, et al. Measuring DNA modifications with the comet assay: a compendium of protocols. Nat Protoc. 2023;18(3):929-89.
• 12. Woods JA, O'Leary KA, McCarthy RP, O'Brien NM. Preservation of comet assay slides: comparison with fresh slides. Mutat Res. 1999;429(2):181-7.
• 13. Huang Q, Fu WL. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin Chem Lab Med. 2005;43(8):841-2.