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Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms

The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms...

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Autores principales: Venturi, Giulia, Zacchini, Federico, Vaccari, Cinzia Lucia, Trerè, Davide, Montanaro, Lorenzo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8691633/
https://www.ncbi.nlm.nih.gov/pubmed/34932578
http://dx.doi.org/10.1371/journal.pone.0261476
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author Venturi, Giulia
Zacchini, Federico
Vaccari, Cinzia Lucia
Trerè, Davide
Montanaro, Lorenzo
author_facet Venturi, Giulia
Zacchini, Federico
Vaccari, Cinzia Lucia
Trerè, Davide
Montanaro, Lorenzo
author_sort Venturi, Giulia
collection PubMed
description The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms of 5.8S rRNA: one more abundant called 5.8S short, one called 5.8S long bearing 5 extra-nucleotides at its 5’ end and one 10 nucleotide shorter called 5.8S cropped. So far, little is known about 5.8S long specific role in cell biology and its function in human pathology. The lack of studies on the three 5.8S isoforms could be due to the techniques usually applied to study ribosome biogenesis, such as Northern blot with radioactively labelled probes, that require strict protective measures, and abundant and high-quality samples. To overcome this issue, we optimized a method that combines primer extension with a fluorescently labeled reverse primer designed on the 3’ of 5.8S rRNA sequence and fragment analysis. The resulting electropherogram shows the peaks corresponding to the three isoforms of 5.8S rRNA. The estimation of the area underneath the peaks allows to directly quantify the isoforms and to express their relative abundance. The relative abundance of 5.8S long and 5.8S short remains constant using scalar dilution of RNA and in samples subjected to partial degradation. 5.8S cropped abundance varies significantly in lower concentrate RNA samples. This method allows to analyze rapidly and safely the abundance of 5.8S rRNA isoforms in samples that have been so far considered not suitable such as poorly concentrated samples, RNA derived from frozen tissue or unique samples.
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spelling pubmed-86916332021-12-22 Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms Venturi, Giulia Zacchini, Federico Vaccari, Cinzia Lucia Trerè, Davide Montanaro, Lorenzo PLoS One Research Article The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms of 5.8S rRNA: one more abundant called 5.8S short, one called 5.8S long bearing 5 extra-nucleotides at its 5’ end and one 10 nucleotide shorter called 5.8S cropped. So far, little is known about 5.8S long specific role in cell biology and its function in human pathology. The lack of studies on the three 5.8S isoforms could be due to the techniques usually applied to study ribosome biogenesis, such as Northern blot with radioactively labelled probes, that require strict protective measures, and abundant and high-quality samples. To overcome this issue, we optimized a method that combines primer extension with a fluorescently labeled reverse primer designed on the 3’ of 5.8S rRNA sequence and fragment analysis. The resulting electropherogram shows the peaks corresponding to the three isoforms of 5.8S rRNA. The estimation of the area underneath the peaks allows to directly quantify the isoforms and to express their relative abundance. The relative abundance of 5.8S long and 5.8S short remains constant using scalar dilution of RNA and in samples subjected to partial degradation. 5.8S cropped abundance varies significantly in lower concentrate RNA samples. This method allows to analyze rapidly and safely the abundance of 5.8S rRNA isoforms in samples that have been so far considered not suitable such as poorly concentrated samples, RNA derived from frozen tissue or unique samples. Public Library of Science 2021-12-21 /pmc/articles/PMC8691633/ /pubmed/34932578 http://dx.doi.org/10.1371/journal.pone.0261476 Text en © 2021 Venturi et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Venturi, Giulia
Zacchini, Federico
Vaccari, Cinzia Lucia
Trerè, Davide
Montanaro, Lorenzo
Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms
title Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms
title_full Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms
title_fullStr Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms
title_full_unstemmed Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms
title_short Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms
title_sort primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8s rrna isoforms
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8691633/
https://www.ncbi.nlm.nih.gov/pubmed/34932578
http://dx.doi.org/10.1371/journal.pone.0261476
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