核酸荧光染料在单细胞凝胶电泳中的染色比较

Comparison of 4 kinds of nucleic acid fluorescent dyes in single-cell gel electrophoresis assay

目的探讨4种核酸染料(EB、SYBR GreenⅠ、Gold View和AO)在单细胞凝胶电泳(SCGE)中的染色特性,以分析用新型核酸荧光染料代替EB染料的可能性。方法分离提取健康人外周血淋巴细胞,分别用0、20、40、60、80μg/m L的H2O2进行处理,中性SCGE检测淋巴细胞DNA损伤情况;分别用EB、SYBR GreenⅠ、Gold View和AO进行染色,荧光显微镜下观察并比较染色结果。分析尾部DNA百分含量(%Tail DNA)以比较组间差异。结果 SCGE结果表明,SYBR GreenⅠ、Gold View和AO均能很好的反应实验结果,不同浓度中,1×~5×SYBR GreenⅠ、2×~5×Gold View、2~5μg/m L AO为最佳染色浓度范围。EB、SYBR GreenⅠ、Gold View和AO的回归系数分别为2.71、2.81、2.73、2.75,其染色效果较为一致,稳定性分析结果表明,3组细胞在48 h以内结果稳定,%Tail DNA差异无统计学意义(P均>0.05);SYBR GreenⅠ、Gold View和AO实验室内精密度分别为6.92%、7.10%、8.25%,优于EB染色(8.35%);SYBR GreenⅠ和Gold View重复性分别为3.07%、2.74%,优于EB染色(3.59%)。结论SYBR GreenⅠ、Gold View和AO均可用于本实验染色,Gold View是更适合用于SCGE中替代EB的核酸荧光染料。

Objective To investigate the DNA staining efficiencies of 4 kinds of nucleic acid fluorescent dyes,including EB,SYBR Green Ⅰ,Gold View and AO,in single-cell gel electrophoresis（ SCGE） assay,and the possibility to use a new nucleic acid fluorescent dye instead of EB. Methods The peripheral blood lymphocytes from healthy individuals were isolated and treated with 0,20,40,60 and 80 μg/m L of H2 O2,respectively. Then,the DNA damages of lymphocytes were detected by the neutral SCGE assay. The DNA was stained with EB,SYBR Green Ⅰ,Gold View and AO dyes,respectively,and the staining results were observed and compared under a fluorescence microscope. In addition,the percentages of tail DNA（ % Tail DNA） from different staining methods were analyzed and compared. Results The results of SCGE showed that SYBR Green Ⅰ,Gold View and AO staining could well reflect the DNA damages of lymphocytes,and that the optimal concentrations for SYBR Green Ⅰ,Gold View and AO were 1 ×-5 ×,2 ×-5 ×and 2-5 μg/m L,respectively. The regression coefficients for EB,SYBR Green Ⅰ,Gold View and AO were 2. 71,2. 81,2. 73 and2. 75,respectively,which indicated that there was consistent dyeing effect between them. The results of % Tail DNA were stable within 48 hours,and there was no significant difference between the 4 fluoresent dyes（ P 0. 05）. The inter-assay coefficients of variation（ CVs） of SYBR Green I,Gold View and AO were 6. 92%,7. 10% and 8. 25%,respectively,which were superior to that of EB（ 8. 35%）. The intra-assay CVs of SYBR Green I and Gold View were 3. 07% and 2. 74%,respectively,which were superior to that of EB（ 3. 59%）. Conclusion SYBR Green I,Gold View and AO may be used for the nucleic acid fluorescent staining,and especially Gold View is more suitable for instead of EB in SCGE.