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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| Format: | Article |
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MDPI AG
2022-11-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/11/22/3578 |
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| author | Artemii A. Ivanov Olga N. Leonova Daniil S. Wiebe Alexsandr V. Krutko Mariya M. Gridina Veniamin S. Fishman Yurii S. Aulchenko Yakov A. Tsepilov Tatiana S. Golubeva |
| author_facet | Artemii A. Ivanov Olga N. Leonova Daniil S. Wiebe Alexsandr V. Krutko Mariya M. Gridina Veniamin S. Fishman Yurii S. Aulchenko Yakov A. Tsepilov Tatiana S. Golubeva |
| author_sort | Artemii A. Ivanov |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T18:25:39Z |
| format | Article |
| id | doaj.art-b39cbcd1663b4196a08574c408f2542a |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-09T18:25:39Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-b39cbcd1663b4196a08574c408f2542a2023-11-24T07:57:42ZengMDPI AGCells2073-44092022-11-011122357810.3390/cells11223578Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle HomogenizationArtemii A. Ivanov0Olga N. Leonova1Daniil S. Wiebe2Alexsandr V. Krutko3Mariya M. Gridina4Veniamin S. Fishman5Yurii S. Aulchenko6Yakov A. Tsepilov7Tatiana S. Golubeva8Institute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, RussiaThe 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.https://www.mdpi.com/2073-4409/11/22/3578cartilage RNA isolationRNA-seqRINintervertebral disc |
| spellingShingle | Artemii A. Ivanov Olga N. Leonova Daniil S. Wiebe Alexsandr V. Krutko Mariya M. Gridina Veniamin S. Fishman Yurii S. Aulchenko Yakov A. Tsepilov Tatiana S. Golubeva Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization Cells cartilage RNA isolation RNA-seq RIN intervertebral disc |
| 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 | cartilage RNA isolation RNA-seq RIN intervertebral disc |
| url | https://www.mdpi.com/2073-4409/11/22/3578 |
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