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

Establishment and optimization of non-coding small RNA detection methods by biotin-labeled probe-liquid hybridization-non-denaturing PAGE

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sRNAU6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sRNAU6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,10μmol/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5μgRNA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）-conjugated 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 μmol/L.There was no significant difference of hybrid chains,acquired by water bath or polymerase chain reaction（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 A549,while the abundance of miRNA-145 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.