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RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules
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 a...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3488209/ https://www.ncbi.nlm.nih.gov/pubmed/22833606 http://dx.doi.org/10.1093/nar/gks698 |
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author | Dong, Zhi-Wei Shao, Peng Diao, Li-Ting Zhou, Hui Yu, Chun-Hong Qu, Liang-Hu |
author_facet | Dong, Zhi-Wei Shao, Peng Diao, Li-Ting Zhou, Hui Yu, Chun-Hong Qu, Liang-Hu |
author_sort | Dong, Zhi-Wei |
collection | PubMed |
description | 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). |
format | Online Article Text |
id | pubmed-3488209 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-34882092012-11-06 RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules Dong, Zhi-Wei Shao, Peng Diao, Li-Ting Zhou, Hui Yu, Chun-Hong Qu, Liang-Hu Nucleic Acids Res Methods Online 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). Oxford University Press 2012-11 2012-07-23 /pmc/articles/PMC3488209/ /pubmed/22833606 http://dx.doi.org/10.1093/nar/gks698 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Dong, Zhi-Wei Shao, Peng Diao, Li-Ting Zhou, Hui Yu, Chun-Hong Qu, Liang-Hu RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules |
title | RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules |
title_full | RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules |
title_fullStr | RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules |
title_full_unstemmed | RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules |
title_short | RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules |
title_sort | rtl-p: a sensitive approach for detecting sites of 2′-o-methylation in rna molecules |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3488209/ https://www.ncbi.nlm.nih.gov/pubmed/22833606 http://dx.doi.org/10.1093/nar/gks698 |
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