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A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone
Molecular differences between cortical and trabecular bone, of relevance to understanding the pathophysiological basis of bone diseases, can be determined only through effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, which combines bone pulverization and homog...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9065961/ https://www.ncbi.nlm.nih.gov/pubmed/35506206 http://dx.doi.org/10.1098/rsob.210387 |
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author | Faraldi, Martina Mangiavini, Laura Conte, Caterina Banfi, Giuseppe Napoli, Nicola Lombardi, Giovanni |
author_facet | Faraldi, Martina Mangiavini, Laura Conte, Caterina Banfi, Giuseppe Napoli, Nicola Lombardi, Giovanni |
author_sort | Faraldi, Martina |
collection | PubMed |
description | Molecular differences between cortical and trabecular bone, of relevance to understanding the pathophysiological basis of bone diseases, can be determined only through effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, which combines bone pulverization and homogenization to extract simultaneously total RNA and proteins from human cortical and trabecular bone from the same carrot. RNA integrity and purity were determined as the 260/280 nm and 260/230 nm absorbance ratios and the 28S/18S rRNA ratio. Protein integrity and quality were evaluated by Coomassie blue staining. Reverse transcription quantitative polymerase chain reaction and immunoblotting for bone-specific genes and proteins were performed to verify the suitability of the isolated material in downstream applications. The 260/280 nm and 260/230 nm absorbance ratios were, on average, less than or equal to 1.8. Bands on agarose gel were consistent with intact RNA, with mean 28S/18S ratios of 1.68 ± 0.35 and 1.88 ± 0.10 for cortical and trabecular bone, respectively. Band patterns after Coomassie blue staining confirmed protein integrity. Successful gene and protein expression analysis, with relevant differences between the two compartments, highlighted the suitability of the material in downstream applications. The method presented here is appropriate and effective for the study of human bone. |
format | Online Article Text |
id | pubmed-9065961 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | The Royal Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-90659612022-05-09 A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone Faraldi, Martina Mangiavini, Laura Conte, Caterina Banfi, Giuseppe Napoli, Nicola Lombardi, Giovanni Open Biol Research Molecular differences between cortical and trabecular bone, of relevance to understanding the pathophysiological basis of bone diseases, can be determined only through effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, which combines bone pulverization and homogenization to extract simultaneously total RNA and proteins from human cortical and trabecular bone from the same carrot. RNA integrity and purity were determined as the 260/280 nm and 260/230 nm absorbance ratios and the 28S/18S rRNA ratio. Protein integrity and quality were evaluated by Coomassie blue staining. Reverse transcription quantitative polymerase chain reaction and immunoblotting for bone-specific genes and proteins were performed to verify the suitability of the isolated material in downstream applications. The 260/280 nm and 260/230 nm absorbance ratios were, on average, less than or equal to 1.8. Bands on agarose gel were consistent with intact RNA, with mean 28S/18S ratios of 1.68 ± 0.35 and 1.88 ± 0.10 for cortical and trabecular bone, respectively. Band patterns after Coomassie blue staining confirmed protein integrity. Successful gene and protein expression analysis, with relevant differences between the two compartments, highlighted the suitability of the material in downstream applications. The method presented here is appropriate and effective for the study of human bone. The Royal Society 2022-05-04 /pmc/articles/PMC9065961/ /pubmed/35506206 http://dx.doi.org/10.1098/rsob.210387 Text en © 2022 The Authors. https://creativecommons.org/licenses/by/4.0/Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, provided the original author and source are credited. |
spellingShingle | Research Faraldi, Martina Mangiavini, Laura Conte, Caterina Banfi, Giuseppe Napoli, Nicola Lombardi, Giovanni A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone |
title | A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone |
title_full | A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone |
title_fullStr | A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone |
title_full_unstemmed | A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone |
title_short | A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone |
title_sort | novel methodological approach to simultaneously extract high-quality total rna and proteins from cortical and trabecular bone |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9065961/ https://www.ncbi.nlm.nih.gov/pubmed/35506206 http://dx.doi.org/10.1098/rsob.210387 |
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