In situ detection of mature microRNAs by labeled extension on ultramer templates.
In situ detection of mature microRNAs by labeled extension on ultramer templates.
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DOI：
10.2144/000113068
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发表时间：
2009-02
期刊：
影响因子：
2.7
通讯作者：
Schmittgen T
中科院分区：
文献类型：
--
作者：
Nuovo G;Lee EJ;Lawler S;Godlewski J;Schmittgen T
我们描述了一种在福尔马林固定石蜡包埋组织中原位检测成熟microRNA （miRNA）的新方法。该方法包括将标记的miRNA延伸杂交到大约100个核苷酸长的超分子模板上，该模板在其3 '端含有miRNA的互补序列。组织的预处理包括用蛋白酶孵育，使基因组DNA暴露于DNA酶消化，从而消除石蜡包埋组织中固有的超聚合物独立DNA合成过程。通过与实时逆转录酶(RT) -PCR、RT原位PCR和使用锁定核酸（LNA）探针的标准原位杂交的直接比较，可以明显看出，超细延伸法只检测成熟的miRNA，更容易优化，结果通常更强，而且比目前使用的LNA探针方法便宜得多。
We describe a new method for the in situ detection of a mature microRNA (miRNA) in formalin-fixed, paraffin-embedded tissues. The method involves the labeled extension of miRNA hybridized to an approximately 100-nucleotide–long ultramer template containing the complementary sequence of the miRNA at its 3′ terminus. Pretreatment of the tissue involves incubation with protease to expose the genomic DNA to DNase digestion, thereby eliminating the ultramer-independent DNA synthesis process inherent in paraffin-embedded tissue. By direct comparison with real-time reverse transcriptase (RT)–PCR, RT in situ PCR, and standard in situ hybridization using a locked nucleic acid (LNA) probe, it was evident that the ultramer extension method detects only the mature miRNA, is easier to optimize, results generally in a stronger signal, and is much less expensive than the LNA probe method currently used.