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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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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. |
format | Online Article Text |
id | pubmed-8691633 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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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