Cargando…
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...
Autores principales: | , , , , , , , , |
---|---|
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 |
_version_ | 1784836298454859776 |
---|---|
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. |
format | Online Article Text |
id | pubmed-9688557 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT ivanovartemiia methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT leonovaolgan methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT wiebedaniils methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT krutkoalexsandrv methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT gridinamariyam methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT fishmanveniamins methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT aulchenkoyuriis methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT tsepilovyakova methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization AT golubevatatianas methodfortheisolationofrnaseqqualityrnafromhumanintervertebraldiscsaftermortarandpestlehomogenization |