Heath, P.R., Appleby-Mallinder, C., Wyles, M.D. et al. (3 more authors) (2016) Expression microdissection isolation of enriched cell populations from archival brain tissue. Journal of Neuroscience Methods. ISSN 0165-0270
Abstract
BACKGROUND: Laser capture microdissection (LCM) is an established technique for the procurement of enriched cell populations that can undergo further downstream analysis, although it does have limitations. Expression microdissection (xMD) is a new technique that begins to address these pitfalls, such as operator dependence and contamination.
NEW METHOD: xMD utilises immunohistochemistry in conjunction with a chromogen to isolate specific cell types by extending the fundamental principles of LCM to create an operator-independent method for the procurement of specific CNS cell types.
RESULTS: We report how xMD enables the isolation of specific cell populations, namely neurones and astrocytes, from rat formalin fixed-paraffin embedded (FFPE) tissue. Subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirms the enrichment of these specific populations. RIN values after xMD indicate samples are sufficient to carry out further analysis.
COMPARISON WITH EXISTING METHOD: xMD offers a rapid method of isolating specific CNS cell types without the need for identification by an operator, reducing the amount of unintentional contamination caused by operator error, whilst also significantly reducing the time required by the current basic LCM technique.
CONCLUSIONS: xMD is a superior method for the procurement of enriched cell populations from post-mortem tissue, which can be utilised to create transcriptome profiles, aiding our understanding of the contribution of these cells to a range of neurological diseases. xMD also addresses the issues associated with LCM, such as reliance on an operator to identify target cells, which can cause contamination, as well as addressing the time consuming nature of LCM.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2016 Elsevier. This is an author produced version of a paper subsequently published in Journal of Neuroscience Methods. Uploaded in accordance with the publisher's self-archiving policy. Article available under the terms of the CC-BY-NC-ND licence (https://creativecommons.org/licenses/by-nc-nd/4.0/) |
Keywords: | Astrocytes; Expression microdissection; Laser capture microdissection; Neurones; RNA; RNA integrity number |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Department of Neuroscience (Sheffield) The University of Sheffield > Sheffield Teaching Hospitals |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 01 Aug 2016 08:58 |
Last Modified: | 18 Jul 2017 05:28 |
Published Version: | http://dx.doi.org/10.1016/j.jneumeth.2016.05.007 |
Status: | Published |
Publisher: | Elsevier |
Refereed: | Yes |
Identification Number: | 10.1016/j.jneumeth.2016.05.007 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:99547 |