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MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs

BACKGROUND: Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature...

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Autores principales: Song, Changnian, Fang, Jinggui, Wang, Chen, Guo, Lei, Nicholas, Kibet Korir, Ma, Zhengqiang
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881865/
https://www.ncbi.nlm.nih.gov/pubmed/20539756
http://dx.doi.org/10.1371/journal.pone.0010861
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author Song, Changnian
Fang, Jinggui
Wang, Chen
Guo, Lei
Nicholas, Kibet Korir
Ma, Zhengqiang
author_facet Song, Changnian
Fang, Jinggui
Wang, Chen
Guo, Lei
Nicholas, Kibet Korir
Ma, Zhengqiang
author_sort Song, Changnian
collection PubMed
description BACKGROUND: Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs. METHODS: In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5′ and 3′ miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5′ RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA. RESULTS: The efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. CONCLUSION: The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor.
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spelling pubmed-28818652010-06-10 MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs Song, Changnian Fang, Jinggui Wang, Chen Guo, Lei Nicholas, Kibet Korir Ma, Zhengqiang PLoS One Research Article BACKGROUND: Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs. METHODS: In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5′ and 3′ miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5′ RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA. RESULTS: The efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. CONCLUSION: The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor. Public Library of Science 2010-06-07 /pmc/articles/PMC2881865/ /pubmed/20539756 http://dx.doi.org/10.1371/journal.pone.0010861 Text en Song et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Song, Changnian
Fang, Jinggui
Wang, Chen
Guo, Lei
Nicholas, Kibet Korir
Ma, Zhengqiang
MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
title MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
title_full MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
title_fullStr MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
title_full_unstemmed MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
title_short MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
title_sort mir-race, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (poncirus trifoliata) micrornas
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881865/
https://www.ncbi.nlm.nih.gov/pubmed/20539756
http://dx.doi.org/10.1371/journal.pone.0010861
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