An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA
Abstract Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (A...
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2020-09-01
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| Series: | JOR Spine |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jsp2.1108 |
| _version_ | 1818991921330126848 |
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| author | Vikrant Piprode Sarthak Mohanty Raffaella Bonavita Sarah Loh Rajakumar Anbazhagan Chandan Saini Robert Pinelli Paul Pricop Chitra L. Dahia |
| author_facet | Vikrant Piprode Sarthak Mohanty Raffaella Bonavita Sarah Loh Rajakumar Anbazhagan Chandan Saini Robert Pinelli Paul Pricop Chitra L. Dahia |
| author_sort | Vikrant Piprode |
| collection | DOAJ |
| description | Abstract Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (AF), and a pair of end plate (EP) that connects the disc to the vertebral bodies. Understanding the molecular and cellular basis of intervertebral disc growth, health, and aging will generate significant information for developing therapeutic approaches. Rapid and efficient preparations of homogeneous and pure cells are crucial for meaningful and rigorous downstream analysis at the cellular, molecular, and biochemical level. Cross‐sample contamination may influence the interpretation of the results. In addition to altering gene expression, slow or delayed isolation procedures will also cause the degradation of cells and biomolecules that create a bias in the outcomes of the study. The mouse model system is extensively used to understand the intervertebral disc biology. Here we describe two protocols: (a) for efficient isolation of pure NP, AF, and EP cells from mouse lumbar intervertebral disc. We validated the purity of the NP and AF cells using ShhCre/+; R26mT/mG/+ dual‐fluorescent reporter mice where all NP cells are GPF+ve, and by the sensitive approach of qPCR analysis using TaqMan probes for Shh, and Brachyury as NP‐specific markers, Tenomodulin as AF‐specific marker, and Osteocalcin as bone‐specific marker. (b) For isolation of high‐quality intact RNA with RIN of 9.3 to 10 from disc cells. These methods will be useful for the rigorous analysis of NP and AF cells, and improve our understanding of intervertebral disc biology. |
| first_indexed | 2024-12-20T20:17:57Z |
| format | Article |
| id | doaj.art-d2f377ce4a444f33a08bd1558b72ef81 |
| institution | Directory Open Access Journal |
| issn | 2572-1143 |
| language | English |
| last_indexed | 2024-12-20T20:17:57Z |
| publishDate | 2020-09-01 |
| publisher | Wiley |
| record_format | Article |
| series | JOR Spine |
| spelling | doaj.art-d2f377ce4a444f33a08bd1558b72ef812022-12-21T19:27:39ZengWileyJOR Spine2572-11432020-09-0133n/an/a10.1002/jsp2.1108An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNAVikrant Piprode0Sarthak Mohanty1Raffaella Bonavita2Sarah Loh3Rajakumar Anbazhagan4Chandan Saini5Robert Pinelli6Paul Pricop7Chitra L. Dahia8Hospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAHospital for Special Surgery New York New York USAAbstract Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (AF), and a pair of end plate (EP) that connects the disc to the vertebral bodies. Understanding the molecular and cellular basis of intervertebral disc growth, health, and aging will generate significant information for developing therapeutic approaches. Rapid and efficient preparations of homogeneous and pure cells are crucial for meaningful and rigorous downstream analysis at the cellular, molecular, and biochemical level. Cross‐sample contamination may influence the interpretation of the results. In addition to altering gene expression, slow or delayed isolation procedures will also cause the degradation of cells and biomolecules that create a bias in the outcomes of the study. The mouse model system is extensively used to understand the intervertebral disc biology. Here we describe two protocols: (a) for efficient isolation of pure NP, AF, and EP cells from mouse lumbar intervertebral disc. We validated the purity of the NP and AF cells using ShhCre/+; R26mT/mG/+ dual‐fluorescent reporter mice where all NP cells are GPF+ve, and by the sensitive approach of qPCR analysis using TaqMan probes for Shh, and Brachyury as NP‐specific markers, Tenomodulin as AF‐specific marker, and Osteocalcin as bone‐specific marker. (b) For isolation of high‐quality intact RNA with RIN of 9.3 to 10 from disc cells. These methods will be useful for the rigorous analysis of NP and AF cells, and improve our understanding of intervertebral disc biology.https://doi.org/10.1002/jsp2.1108developmentgeneticsPreclinical modelsregenerative medicine |
| spellingShingle | Vikrant Piprode Sarthak Mohanty Raffaella Bonavita Sarah Loh Rajakumar Anbazhagan Chandan Saini Robert Pinelli Paul Pricop Chitra L. Dahia An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA JOR Spine development genetics Preclinical models regenerative medicine |
| title | An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA |
| title_full | An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA |
| title_fullStr | An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA |
| title_full_unstemmed | An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA |
| title_short | An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA |
| title_sort | optimized step by step protocol for isolation of nucleus pulposus annulus fibrosus and end plate cells from the mouse intervertebral discs and subsequent preparation of high quality intact total rna |
| topic | development genetics Preclinical models regenerative medicine |
| url | https://doi.org/10.1002/jsp2.1108 |
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