Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality
In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel comple...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-05-01
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| Series: | Frontiers in Neuroscience |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fnins.2023.1198154/full |
| _version_ | 1797824294635438080 |
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| author | Nina Hahn Nina Hahn Martin Bens Marin Kempfer Christin Reißig Lars Schmidl Christian Geis Christian Geis |
| author_facet | Nina Hahn Nina Hahn Martin Bens Marin Kempfer Christin Reißig Lars Schmidl Christian Geis Christian Geis |
| author_sort | Nina Hahn |
| collection | DOAJ |
| description | In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols. |
| first_indexed | 2024-03-13T10:36:50Z |
| format | Article |
| id | doaj.art-395f6758eb2a4ccb846aa023f1755585 |
| institution | Directory Open Access Journal |
| issn | 1662-453X |
| language | English |
| last_indexed | 2024-03-13T10:36:50Z |
| publishDate | 2023-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Neuroscience |
| spelling | doaj.art-395f6758eb2a4ccb846aa023f17555852023-05-18T05:42:14ZengFrontiers Media S.A.Frontiers in Neuroscience1662-453X2023-05-011710.3389/fnins.2023.11981541198154Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining qualityNina Hahn0Nina Hahn1Martin Bens2Marin Kempfer3Christin Reißig4Lars Schmidl5Christian Geis6Christian Geis7Section of Translational Neuroimmunology, Department of Neurology, Jena University Hospital, Jena, GermanyCenter for Sepsis Control and Care, Jena University Hospital, Jena, GermanyLeibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, GermanySection of Translational Neuroimmunology, Department of Neurology, Jena University Hospital, Jena, GermanySection of Translational Neuroimmunology, Department of Neurology, Jena University Hospital, Jena, GermanySection of Translational Neuroimmunology, Department of Neurology, Jena University Hospital, Jena, GermanySection of Translational Neuroimmunology, Department of Neurology, Jena University Hospital, Jena, GermanyCenter for Sepsis Control and Care, Jena University Hospital, Jena, GermanyIn comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols.https://www.frontiersin.org/articles/10.3389/fnins.2023.1198154/fullspatial transcriptomicsVisium spatialmouse brain transcriptomeRNA qualityRNA protectionimmunofluorescent staining |
| spellingShingle | Nina Hahn Nina Hahn Martin Bens Marin Kempfer Christin Reißig Lars Schmidl Christian Geis Christian Geis Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality Frontiers in Neuroscience spatial transcriptomics Visium spatial mouse brain transcriptome RNA quality RNA protection immunofluorescent staining |
| title | Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality |
| title_full | Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality |
| title_fullStr | Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality |
| title_full_unstemmed | Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality |
| title_short | Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality |
| title_sort | protecting rna quality for spatial transcriptomics while improving immunofluorescent staining quality |
| topic | spatial transcriptomics Visium spatial mouse brain transcriptome RNA quality RNA protection immunofluorescent staining |
| url | https://www.frontiersin.org/articles/10.3389/fnins.2023.1198154/full |
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