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[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌组织石蜡切片进行杂交实验建立肺癌荧光原位杂交(fluorescence in situ hybridization,FISH)的检测体系。[结果]RBM5 15℃标记12h可获得合适的探针,探针与样本杂交后样本细胞内出现清晰明亮的绿色荧光信号,通过与呈橘红色荧光信号的CEP-3探针比较,可以判断肺癌细胞是否存在RBM5的缺失。[结论]RBM5探针制备的最佳条件是15℃标记12h,FISH实验的参数为10μg/ml蛋白酶K处理样本100 min、探针与样本37℃杂交16h、2×SSC/0.3%NP-40洗涤杂交样本5min。该实验体系适用于肺癌组织RBM5的FISH检测。
[Objective] The research aimed to study the preparation method of RBM5 (RNA-binding motif protein 5) fluorescent probe and establish the in situ hybridization system of RBM5 fluorescent probe for lung cancer tissue detection. [Method] The plasmid of RP11-493K19 was used as the material, and the plasmid containing RBM5 was extracted for PCR validation. The RBM5 fluorescent probe was prepared by nick translation method. Fluorescent in situ hybridization was established with human lung cancer tissue paraffin sections by fluorescence in situ hybridization hybridization, FISH) detection system. [Result] RBM5 could be labeled with suitable probe at 15 ℃ for 12h. After the hybridization of the probe with the sample, clear and bright green fluorescent signal appeared in the cell and the lung cancer could be judged by comparing with the CEP-3 probe showing the orange fluorescent signal Is there a deletion of RBM5 in the cells? [Conclusion] The optimal conditions for the preparation of RBM5 probe were 12 h at 15 ℃, 10 min at FISH and 100 h at 37 ℃ with 2 × SSC / 0.3% NP-40 Wash the hybridization sample for 5 min. The experimental system is suitable for FISH detection of RBM5 in lung cancer.