RBM5荧光原位杂交探针的制备及应用

Method of fluorescence labeling of RBM5 and its application

[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] To explore the method of labeling RBM5 with fluorescein and establish a technical standard for detecting lung canc- er by in situ hybridization with this RBM5 fluorescence probe. [ Methods] RBM5 was extracted from RPll -493K19 bacteria and con- firmed by PCR. Nick translation was adopted to label RBM5 with Green dUTP and paraffin - embedded tissue specimens of human lung cancer were used to conduct the in situ hybridization test. [ Results ] The suitable labeling program was labeling 12 h under 15℃. There were clear and bright green fluorescent signals in lung cancer cells after in situ hybridization experiment, by comparing with chromosome e- numeration probe CEP- 3 ,the loss of RBM5 could be detected. [ Conclusion] The suitable labeling method of RBM5 was labeling 12 h under 15℃. The optimization model for fluorescence in situ hybridization test was digesting specimen with proteinase K for 100min, hybrid- ization at 37℃ for 16h and washing specimen with 2 × SSC/0. 1% ethoxylated octylphenol for 5min. This program is excellent for the de- tection of lung cancer by FISH.