title: RTL-P: a sensitive approach for detecting sites of 2'-O-methylation in RNA molecules.
abstract: 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).
authors: Key Laboratory of Gene Engineering of the Ministry of Education, Sun Yat-sen University, Guangzhou 510275, PR China.
authors: Zhi-Wei
authors: ZW
authors: Dong
authors: Peng
authors: P
authors: Shao
authors: Li-Ting
authors: LT
authors: Diao
authors: Hui
authors: H
authors: Zhou
authors: Chun-Hong
authors: CH
authors: Yu
authors: Liang-Hu
authors: LH
authors: Qu
mesh_terms: D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics
chemical_list: D012335:RNA, Ribosomal; D058727:RNA, Small Untranslated; D012265:Ribonucleotides; D012313:RNA