Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes -: art. no. e175
Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes -: art. no. e175
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DOI：
10.1093/nar/gnh171
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Havelda, Z
中科院分区：
文献类型：
--
作者：
Válóczi, A;Hornyik, C;Havelda, Z
我们在这里描述了一种新的方法，用于通过使用LNA（锁核酸）修饰的寡核苷酸的北方印迹分析来高效检测microRNA。为了利用LNA与其靶RNA分子的杂交特性，我们设计了几种LNA修饰的寡核苷酸探针用于检测动物和植物中不同的microRNA。通过使用其中每三个核苷酸位置被LNA取代的设计用LNA修饰DNA寡核苷酸，我们可以在采用标准末端标记技术和杂交条件的北方印迹分析中使用探针。与DNA探针相比，通过北方印迹检测成熟microRNA的灵敏度提高了至少10倍，同时具有高度特异性，如通过使用不同的单和双错配LNA探针所证明的。除了作为北方探针高效之外，相同的LNA修饰的寡核苷酸探针还可用于通过LNA寡核苷酸微阵列的miRNA原位杂交和miRNA表达谱分析。
We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.