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Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform

BACKGROUND: The 5′ untranslated regions of mRNA play an important role in their translation. RESULTS: Here, we describe the development of four methods of profiling mRNA 5′ ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5′ end of RNA Transcript)...

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Detalles Bibliográficos
Autores principales: Machida, Ryuji J., Lin, Ya-Ying
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4086933/
https://www.ncbi.nlm.nih.gov/pubmed/25003736
http://dx.doi.org/10.1371/journal.pone.0101812
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author Machida, Ryuji J.
Lin, Ya-Ying
author_facet Machida, Ryuji J.
Lin, Ya-Ying
author_sort Machida, Ryuji J.
collection PubMed
description BACKGROUND: The 5′ untranslated regions of mRNA play an important role in their translation. RESULTS: Here, we describe the development of four methods of profiling mRNA 5′ ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5′ end of RNA Transcript) technology, while the second involves replacing the 5′ cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5′ end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5′ end. CONCLUSIONS: These methods are suitable for global mapping of mRNA 5′ ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure.
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spelling pubmed-40869332014-07-14 Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform Machida, Ryuji J. Lin, Ya-Ying PLoS One Research Article BACKGROUND: The 5′ untranslated regions of mRNA play an important role in their translation. RESULTS: Here, we describe the development of four methods of profiling mRNA 5′ ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5′ end of RNA Transcript) technology, while the second involves replacing the 5′ cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5′ end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5′ end. CONCLUSIONS: These methods are suitable for global mapping of mRNA 5′ ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure. Public Library of Science 2014-07-08 /pmc/articles/PMC4086933/ /pubmed/25003736 http://dx.doi.org/10.1371/journal.pone.0101812 Text en © 2014 Machida, Lin 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
Machida, Ryuji J.
Lin, Ya-Ying
Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform
title Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform
title_full Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform
title_fullStr Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform
title_full_unstemmed Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform
title_short Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform
title_sort four methods of preparing mrna 5′ end libraries using the illumina sequencing platform
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4086933/
https://www.ncbi.nlm.nih.gov/pubmed/25003736
http://dx.doi.org/10.1371/journal.pone.0101812
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