摘要
目的比较两种不同荧光标记的PIK3CA siRNA体外转染特性，筛选出转染效率高、抗淬灭能力强的PIK3CA siRNA。方法利用脂质体LJpofectamine 2000转染两种荧光标记PIK3CA siRNA及阴性对照siRNA人胃癌细胞BGC－823，倒置荧光显微镜观察其荧光分布和淬灭情况，并通过ImageJ软件分析其转染效率。实时定量PCR检测不同荧光标记的PIK3C AsiRNA对靶mRNA表达的影响。结果相同转染条件下，两种荧光标记的PIK3CA siRNA转染效率及抗淬灭能力不同。Cy3或FAM标记的PIK3CA siRNA与阴性对照siRNA转染效率无明显差异，但Cy3标记的PIK3CA siRNA与阴性对照siRNA转染效率较FAM标记的明显升高（P〈0．05）。Cy3标记的PIK3CA siRNA对靶mRNA的抑制作用明显高于FAM标记的PIK3CA siRNA（p〈0．05）。结论Cy3标记PIK3CA siRNA可以起到良好的示踪作用，为进一步构建Cy3标记PIK3CA siRNA纳米粒子提供了重要的实验依据。
Objective To compare transfection properties of two different fluorescently labeled PIK3CA siRNA and to screen out PIK3CA siRNA with high transfection efficiency and strong antiquenching ability. Methods Two different fluorescently labeled PIK3CA siRNA was transfected into gastric cancer cell BGC-823 by Lipofectamine 2000. The distribution and quenching of fluorescence were observed by inverted fluorescence microscope. Image J soft- ware was used to analyze their transfeetion efficiency. Real-time quantitative PCR was performed to detect the effect of different fluorescently labeled PIK3CA siRNA on PIK3CA mRNA expression. Results The transfection efficiency and anti-quenching ability of two different fluoreseently labeled PIK3 CA siRNA were different under the same transfection conditions. The transfection efficiency showed no significant differences between Cy3 or FAM labeled PIK3CA siRNA and negative control siRNA（ P 〉 0.05 ） , but the transfection efficiency of Cy3 labeled PIK3CA siRNA and negative control siRNA was significantly higher than the FAM labeled （P 〈 0.05 ）. Inhibitory efficacy of target mRNA expression induced by Cy3 labeled PIK3CA siRNA was significantly higher than that of FAM labeled PIK3CA siRNA. Conclusion Cy3 labeled PIK3CA siRNA could act as a good tracer and provide an important evidence for further construction of Cy3 labeled PIK3CA siRNA nanopartiele.
出处
《中国基层医药》
CAS
2013年第5期644-646,共3页
Chinese Journal of Primary Medicine and Pharmacy
基金
广东省广州市教育局资助项目（10A186）
广州市卫生局资助项目（201102A213006）
广东省卫生厅资助项目（A2011290）
关键词
PIK3CA基因
荧光标记siRNA
转染
PIK3 CA gene
Fluorescently labeled small interfering RNA
Transfection
