Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization
The isolation of high‐quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate‐phenol‐chloroform ext...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2018-06-01
|
| Series: | JOR Spine |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jsp2.1017 |
| _version_ | 1819038458279100416 |
|---|---|
| author | Stephanie Caprez Ursula Menzel Zhen Li Sibylle Grad Mauro Alini Marianna Peroglio |
| author_facet | Stephanie Caprez Ursula Menzel Zhen Li Sibylle Grad Mauro Alini Marianna Peroglio |
| author_sort | Stephanie Caprez |
| collection | DOAJ |
| description | The isolation of high‐quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate‐phenol‐chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on‐column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real‐time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans. |
| first_indexed | 2024-12-21T08:37:38Z |
| format | Article |
| id | doaj.art-a37a960d017545b0bb09c573e730057b |
| institution | Directory Open Access Journal |
| issn | 2572-1143 |
| language | English |
| last_indexed | 2024-12-21T08:37:38Z |
| publishDate | 2018-06-01 |
| publisher | Wiley |
| record_format | Article |
| series | JOR Spine |
| spelling | doaj.art-a37a960d017545b0bb09c573e730057b2022-12-21T19:10:02ZengWileyJOR Spine2572-11432018-06-0112n/an/a10.1002/jsp2.1017Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverizationStephanie Caprez0Ursula Menzel1Zhen Li2Sibylle Grad3Mauro Alini4Marianna Peroglio5AO Research Institute Davos Davos SwitzerlandAO Research Institute Davos Davos SwitzerlandAO Research Institute Davos Davos SwitzerlandAO Research Institute Davos Davos SwitzerlandAO Research Institute Davos Davos SwitzerlandAO Research Institute Davos Davos SwitzerlandThe isolation of high‐quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate‐phenol‐chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on‐column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real‐time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans.https://doi.org/10.1002/jsp2.1017gene expressionintervertebral discreal‐time PCRRNA isolation |
| spellingShingle | Stephanie Caprez Ursula Menzel Zhen Li Sibylle Grad Mauro Alini Marianna Peroglio Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization JOR Spine gene expression intervertebral disc real‐time PCR RNA isolation |
| title | Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization |
| title_full | Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization |
| title_fullStr | Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization |
| title_full_unstemmed | Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization |
| title_short | Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization |
| title_sort | isolation of high quality rna from intervertebral disc tissue via pronase predigestion and tissue pulverization |
| topic | gene expression intervertebral disc real‐time PCR RNA isolation |
| url | https://doi.org/10.1002/jsp2.1017 |
| work_keys_str_mv | AT stephaniecaprez isolationofhighqualityrnafromintervertebraldisctissueviapronasepredigestionandtissuepulverization AT ursulamenzel isolationofhighqualityrnafromintervertebraldisctissueviapronasepredigestionandtissuepulverization AT zhenli isolationofhighqualityrnafromintervertebraldisctissueviapronasepredigestionandtissuepulverization AT sibyllegrad isolationofhighqualityrnafromintervertebraldisctissueviapronasepredigestionandtissuepulverization AT mauroalini isolationofhighqualityrnafromintervertebraldisctissueviapronasepredigestionandtissuepulverization AT mariannaperoglio isolationofhighqualityrnafromintervertebraldisctissueviapronasepredigestionandtissuepulverization |