RTL-P: a sensitive approach for detecting sites of 2'-O-methylation in RNA molecules.
RTL-P: a sensitive approach for detecting sites of 2'-O-methylation in RNA molecules.
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DOI：
10.1093/nar/gks698
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发表时间：
2012-11-01
影响因子：
14.9
通讯作者：
Qu LH
中科院分区：
文献类型：
--
作者：
Dong ZW;Shao P;Diao LT;Zhou H;Yu CH;Qu LH
2′-O-甲基化存在于各种细胞RNA中，对RNA的生物发生和功能至关重要。已经开发了几种方法来鉴定和定位RNA中的2′-O-甲基化位点;然而，RNA修饰的检测，特别是在低丰度RNA和3′-末端具有2′-O-甲基化的小非编码RNA中，仍然是一项困难的任务。在这里，我们介绍了一种新的方法来检测2′-O-甲基化位点在不同的RNA物种，被称为RTL-P [在低脱氧核糖核苷三磷酸（dNTP）浓度的逆转录聚合酶链反应（PCR）]，表现出精确的映射和上级灵敏度相比，以前的技术。RTL-P的主要步骤包括在低dNTP浓度下通过逆转录酶进行的位点特异性引物延伸和半定量PCR扩增步骤。不需要放射性标记或荧光引物。通过设计特异性RT引物，我们使用RTL-P来检测人类和酵母核糖体RNA（rRNA）以及小鼠piwi相互作用RNA（piRNA）中先前鉴定的和新的2′-O-甲基化位点。这些结果证明了RTL-P用于系统分析不同RNA种类中完全或部分甲基化残基的强大应用，包括低丰度RNA或小的非编码RNA，如piRNA和microRNA（miRNA）。
2′-O-methylation is present within various cellular RNAs and is essential to RNA biogenesis and functionality. Several methods have been developed for the identification and localization of 2′-O-methylated sites in RNAs; however, the detection of RNA modifications, especially in low-abundance RNAs and small non-coding RNAs with a 2′-O-methylation at the 3′-end, remains a difficult task. Here, we introduce a new method to detect 2′-O-methylated sites in diverse RNA species, referred to as RTL-P [Reverse Transcription at Low deoxy-ribonucleoside triphosphate (dNTP) concentrations followed by polymerase chain reaction (PCR)] that demonstrates precise mapping and superior sensitivity compared with previous techniques. The main procedures of RTL-P include a site-specific primer extension by reverse transcriptase at a low dNTP concentration and a semi-quantitative PCR amplification step. No radiolabeled or fluorescent primers are required. By designing specific RT primers, we used RTL-P to detect both previously identified and novel 2′-O-methylated sites in human and yeast ribosomal RNAs (rRNAs), as well as mouse piwi-interacting RNAs (piRNAs). These results demonstrate the powerful application of RTL-P for the systematic analysis of fully or partially methylated residues in diverse RNA species, including low-abundance RNAs or small non-coding RNAs such as piRNAs and microRNAs (miRNAs).
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