结合于红细胞表面单链短核苷酸提取方法学建立研究  

Study on the method to extract short single stranded nucleotidebinding on the surface of the red blood cells

作　　者：张印则[1] 葛伟鹏[2] 吴凡[1] 梁延连[1] 苏宇清[1] 李大成[1] 徐华[3] ZHANG Yinze GE Weipeng WU Fan LIANG Yanlian SU Yuqin LI Dacheng XU Hua(Shenzhen Blood Center, Shenzhen 518035, China Dalian Medical University Shanxi Blood Center.)

机构地区：[1]深圳市血液中心,广东深圳518035 [2]大连医科大学 [3]陕西省血液中心

出　　处：《中国输血杂志》2016年第8期782-784,共3页Chinese Journal of Blood Transfusion

基　　金：深圳市科技计划项目(JCYJ20140403092619633)

摘　　要：目的建立用于红细胞消减SELEX筛选的单链短核苷酸的提取方法。方法使用高灵敏度的硅烷磁珠法提取结合于红细胞表面的单链短核苷酸,并采用实时荧光定量PCR法对其进行定量检测。同时以常用的酚氯仿提取法为对照,评价硅烷磁珠提取法的优劣。结果硅烷磁珠法提取结合于红细胞表面的单链短核苷酸的拷贝数为(4.79±0.13)×10^(10)/μL,而常用的酚氯仿法为(3.61±0.14)×10^(10)/μL。两者相比统计学差异显著(t=17.825,P<0.05)。结论与酚氯仿提取法相比,硅烷磁珠法提取ssDNA的灵敏度更高,可获得更多的结合于红细胞表面的单链短核苷酸。Objective To establish a method for the extraction of short single stranded nucleotides binding on the surface of red blood cells generated by cellular based systematic evolution of ligands by exponential enrichment. Methods Silane magnetic beads method was used to extract short single stranded nueleotide bound to the surface of red blood cells. The quantity wasdetected by real-time fluorescence quantitative PCR method. At the same time, the common phenol chloroformextraction method was used as the control. The difference of the two methods was compared and evaluated. Results The result of silane magnetic beads extraction method to extract single stranded short nucleotide binding on the surface of red blood cells was （4.79 ±0. 13） ×10^10 copies/μL, while that of the phenol chloroform extraction method was （3.61 ±0. 14） × 10^10 copies/μL. There was statistically significant difference between the two methods （t = 17. 825, P 〈 0. 05）. Conclusion Compared with phenol chloroform extraction method, the silane magnetic beads extraction method to extract ssDNA had higher sensitivity, and it can obtain more single stranded short nucleotide binding to the surface of red blood cells.

关 键 词：SSDNA 磁珠法 酚氯仿法 荧光定量 

分 类 号：R457.11[医药卫生—治疗学] Q753[医药卫生—临床医学]