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Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization

The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage...

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Autores principales: Ivanov, Artemii A., Leonova, Olga N., Wiebe, Daniil S., Krutko, Alexsandr V., Gridina, Mariya M., Fishman, Veniamin S., Aulchenko, Yurii S., Tsepilov, Yakov A., Golubeva, Tatiana S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9688557/
https://www.ncbi.nlm.nih.gov/pubmed/36429007
http://dx.doi.org/10.3390/cells11223578
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author Ivanov, Artemii A.
Leonova, Olga N.
Wiebe, Daniil S.
Krutko, Alexsandr V.
Gridina, Mariya M.
Fishman, Veniamin S.
Aulchenko, Yurii S.
Tsepilov, Yakov A.
Golubeva, Tatiana S.
author_facet Ivanov, Artemii A.
Leonova, Olga N.
Wiebe, Daniil S.
Krutko, Alexsandr V.
Gridina, Mariya M.
Fishman, Veniamin S.
Aulchenko, Yurii S.
Tsepilov, Yakov A.
Golubeva, Tatiana S.
author_sort Ivanov, Artemii A.
collection PubMed
description The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture. In our study, we used a combination of the traditional QIAzol protocol (Qiagen, Germany) and RNEasy column purification (Qiagen, Germany) to obtain high-quality RNA from post-surgical intervertebral disc fragments. Only a mortar and a pestle were used for grinding, making our method particularly accessible. The isolated RNA with a RIN of ~7 is suitable for studying the expression profile of chondrocytes in situ. RNA-seq analysis of three samples demonstrated cell type ratios to be mostly relevant to intervertebral disc tissues, with over 70% of the chondrocytes of the three subtypes having an admixture of blood-related cells.
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spelling pubmed-96885572022-11-25 Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization Ivanov, Artemii A. Leonova, Olga N. Wiebe, Daniil S. Krutko, Alexsandr V. Gridina, Mariya M. Fishman, Veniamin S. Aulchenko, Yurii S. Tsepilov, Yakov A. Golubeva, Tatiana S. Cells Article The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture. In our study, we used a combination of the traditional QIAzol protocol (Qiagen, Germany) and RNEasy column purification (Qiagen, Germany) to obtain high-quality RNA from post-surgical intervertebral disc fragments. Only a mortar and a pestle were used for grinding, making our method particularly accessible. The isolated RNA with a RIN of ~7 is suitable for studying the expression profile of chondrocytes in situ. RNA-seq analysis of three samples demonstrated cell type ratios to be mostly relevant to intervertebral disc tissues, with over 70% of the chondrocytes of the three subtypes having an admixture of blood-related cells. MDPI 2022-11-11 /pmc/articles/PMC9688557/ /pubmed/36429007 http://dx.doi.org/10.3390/cells11223578 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ivanov, Artemii A.
Leonova, Olga N.
Wiebe, Daniil S.
Krutko, Alexsandr V.
Gridina, Mariya M.
Fishman, Veniamin S.
Aulchenko, Yurii S.
Tsepilov, Yakov A.
Golubeva, Tatiana S.
Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization
title Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization
title_full Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization
title_fullStr Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization
title_full_unstemmed Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization
title_short Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization
title_sort method for the isolation of “rna-seq-quality” rna from human intervertebral discs after mortar and pestle homogenization
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9688557/
https://www.ncbi.nlm.nih.gov/pubmed/36429007
http://dx.doi.org/10.3390/cells11223578
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