生物素标记探针-液相杂交-非变性PAGE检测非编码小RNA方法的建立和优化

摘要

目的 建立生物素标记探针-液相杂交-非变性聚丙烯酰胺凝胶电泳(PAGE)检测非编码小RNA(sRNA)的方法.方法 将生物素标记的sRNA U6寡核苷酸探针经非变性PAGE电泳后转印至尼龙膜上,然后用辣根过氧化物酶(HRP)耦联链霉亲和素进行检测.从细胞中提取总RNA,用已标记U6探针优化液相杂交退火条件及杂交缓冲液;用建立的方法 检测并计算BEAS-2B、A549细胞中sRNA U6与miRNA145的含量.结果 将Biotin-11-dUTP成功标记至寡核苷酸上,随着寡核苷酸浓度增加,检测信号强度越强,10 μmol/L时达到最强.采用水浴反应条件与采用聚合酶链反应(PCR)仪梯次降温反应条件下得到的杂化链无明显差异;以磷酸盐缓冲液作为杂交缓冲液优于Tris-HCl、DEPC水;BEAS-2B、A549细胞5 μg RNA中U6含量接近;BEAS-2B细胞中miRNA145绝对含量高于A549细胞.结论 成功建立并优化了sRNA检测新方法,为简单、易行、可靠地检测sRNA提供了可能.%Objective To develop a method for detecting non-coding small RNA(sRNA) by biotin-labeled probe-liquid hybridization-non-denaturing polyacrylamide gel electrophoresis(PAGE). Methods The biotinylated oligonucleotides of sRNA U6 were treated by non-denaturing PAGE,transferring to nylon membrane,and then were detected by horseradish peroxidase(HRP)-conju-gated streptavidin-biotin. U6 and miRNA-145 contents in BEAS-2B and A549 cells were detected and calculated through the established method. Results The U6 oligonucleotides were successfully labeled with bio-11-dUTP, which had the strongest signal at concentration of 10 μtmol/L. There was no significant difference of hybrid chains,acquired by water bath or polymerase chain reac-tion(PCR) instrument. According to liquid hybridization solution,phosphate buffer was superior to Tris-HCl and DEPC water. U6 content in BEAS-2B cells was almost equivalent to the content in A54 9,while the abundance of miRNA-14 5 in BEAS-2B was higher than that in A549. Conclusion A new method for detecting sRNA was successfully established and optimized, which could offer possibility for simple and reliable detection of sRNA.