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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 single stranded short stranded RNA for SELEC screening of erythrocytes. Methods Single-stranded short-stranded nucleotides that bound to the surface of erythrocytes were extracted by high-sensitivity silane-magnetic bead method and detected by real-time fluorescence quantitative PCR. At the same time, the common phenol-chloroform extraction method was used as a control to evaluate the advantages and disadvantages of silane bead extraction. RESULTS: The number of single-stranded short stranded DNA bound to the surface of erythrocytes was (4.79 ± 0.13) × 10 10 / μL, while the common phenol-chloroform method was (3.61 ± 0.14) × 10 ~ (10) / μL. The difference between the two groups was statistically significant (t = 17.825, P <0.05). Conclusion Compared with the phenol-chloroform extraction method, the sensitivity of silane-based magnetic beads extraction ssDNA is higher, and more single-stranded short stranded nucleotides can be obtained that bind to the surface of erythrocytes.