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Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR
Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2′-hydroxyl-group of ribonucleotides (2′-O-methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an effi...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191718/ https://www.ncbi.nlm.nih.gov/pubmed/30076204 http://dx.doi.org/10.1261/rna.065144.117 |
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author | Wang, Nan Qu, Shuang Sun, Wu Zeng, Ziyi Liang, Hongwei Zhang, Chen-Yu Chen, Xi Zen, Ke |
author_facet | Wang, Nan Qu, Shuang Sun, Wu Zeng, Ziyi Liang, Hongwei Zhang, Chen-Yu Chen, Xi Zen, Ke |
author_sort | Wang, Nan |
collection | PubMed |
description | Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2′-hydroxyl-group of ribonucleotides (2′-O-methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an efficient method for quantifying small RNA 3′ terminal 2′-O-methylation, it is difficult to monitor the dynamic change of 3′ terminal 2′-O-methylation during various biological processes. Capitalizing on the finding that 3′ terminal RNA 2′-O-methylation can inhibit the activity of poly(A) polymerase, an enzyme that can add the poly(A)-tail to RNA, we develop a method by which the 2′-O-methylation level of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), can be directly quantified based on the poly(A)-tailed RT-qPCR technique. With this method, we successfully determine the 2′-O-methylation level of miRNAs in Arabidopsis thaliana and mouse lung tissue, piRNA in human seminal plasma, and monitor the alteration of miRNA 2′-O-methylation in Drosophila Schneider 2 cells after knockdown of Drosophila methyltransferase protein Hua enhancer 1 (DmHen-1). |
format | Online Article Text |
id | pubmed-6191718 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-61917182019-11-01 Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR Wang, Nan Qu, Shuang Sun, Wu Zeng, Ziyi Liang, Hongwei Zhang, Chen-Yu Chen, Xi Zen, Ke RNA Article Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2′-hydroxyl-group of ribonucleotides (2′-O-methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an efficient method for quantifying small RNA 3′ terminal 2′-O-methylation, it is difficult to monitor the dynamic change of 3′ terminal 2′-O-methylation during various biological processes. Capitalizing on the finding that 3′ terminal RNA 2′-O-methylation can inhibit the activity of poly(A) polymerase, an enzyme that can add the poly(A)-tail to RNA, we develop a method by which the 2′-O-methylation level of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), can be directly quantified based on the poly(A)-tailed RT-qPCR technique. With this method, we successfully determine the 2′-O-methylation level of miRNAs in Arabidopsis thaliana and mouse lung tissue, piRNA in human seminal plasma, and monitor the alteration of miRNA 2′-O-methylation in Drosophila Schneider 2 cells after knockdown of Drosophila methyltransferase protein Hua enhancer 1 (DmHen-1). Cold Spring Harbor Laboratory Press 2018-11 /pmc/articles/PMC6191718/ /pubmed/30076204 http://dx.doi.org/10.1261/rna.065144.117 Text en © 2018 Wang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Article Wang, Nan Qu, Shuang Sun, Wu Zeng, Ziyi Liang, Hongwei Zhang, Chen-Yu Chen, Xi Zen, Ke Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR |
title | Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR |
title_full | Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR |
title_fullStr | Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR |
title_full_unstemmed | Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR |
title_short | Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR |
title_sort | direct quantification of 3′ terminal 2′-o-methylation of small rnas by rt-qpcr |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191718/ https://www.ncbi.nlm.nih.gov/pubmed/30076204 http://dx.doi.org/10.1261/rna.065144.117 |
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