Comparison of 4 kinds of nucleic acid fluorescent dyes in single-cell gel electrophoresis assay / 临床检验杂志

ABSTRACT

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/mL of H2O2,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/mL,respectively.The regression coefficients for EB,SYBR Green Ⅰ,Gold View and AO were 2.71,2.81,2.73 and 2.75,respectively,which indicated that there was consistent dyeing effect between them.The results of ％ Tail DNA were stable with in 48 hours,and there was no significant difference between the 4 fluoresent dyes (P ＞ O.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.