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A Novel HPLC-Based Method to Investigate on RNA after Fixation

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the app...

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Autores principales: Fattorini, Paolo, Forzato, Cristina, Tierno, Domenico, De Martino, Eleonora, Azzalini, Eros, Canzonieri, Vincenzo, Stanta, Giorgio, Bonin, Serena
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588918/
https://www.ncbi.nlm.nih.gov/pubmed/33066070
http://dx.doi.org/10.3390/ijms21207540
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author Fattorini, Paolo
Forzato, Cristina
Tierno, Domenico
De Martino, Eleonora
Azzalini, Eros
Canzonieri, Vincenzo
Stanta, Giorgio
Bonin, Serena
author_facet Fattorini, Paolo
Forzato, Cristina
Tierno, Domenico
De Martino, Eleonora
Azzalini, Eros
Canzonieri, Vincenzo
Stanta, Giorgio
Bonin, Serena
author_sort Fattorini, Paolo
collection PubMed
description RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.
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spelling pubmed-75889182020-10-29 A Novel HPLC-Based Method to Investigate on RNA after Fixation Fattorini, Paolo Forzato, Cristina Tierno, Domenico De Martino, Eleonora Azzalini, Eros Canzonieri, Vincenzo Stanta, Giorgio Bonin, Serena Int J Mol Sci Article RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation. MDPI 2020-10-13 /pmc/articles/PMC7588918/ /pubmed/33066070 http://dx.doi.org/10.3390/ijms21207540 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Fattorini, Paolo
Forzato, Cristina
Tierno, Domenico
De Martino, Eleonora
Azzalini, Eros
Canzonieri, Vincenzo
Stanta, Giorgio
Bonin, Serena
A Novel HPLC-Based Method to Investigate on RNA after Fixation
title A Novel HPLC-Based Method to Investigate on RNA after Fixation
title_full A Novel HPLC-Based Method to Investigate on RNA after Fixation
title_fullStr A Novel HPLC-Based Method to Investigate on RNA after Fixation
title_full_unstemmed A Novel HPLC-Based Method to Investigate on RNA after Fixation
title_short A Novel HPLC-Based Method to Investigate on RNA after Fixation
title_sort novel hplc-based method to investigate on rna after fixation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588918/
https://www.ncbi.nlm.nih.gov/pubmed/33066070
http://dx.doi.org/10.3390/ijms21207540
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