Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality

Frontiers in Neuroscience

Neuroscience

Martin Bens¹  Christin Reißig²  Lars Schmidl²  Marin Kempfer²  Nina Hahn³  Christian Geis³ 

[1] Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany;Section of Translational Neuroimmunology, Department of Neurology, Jena University Hospital, Jena, Germany;Section of Translational Neuroimmunology, Department of Neurology, Jena University Hospital, Jena, Germany;Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany;

关键词: spatial transcriptomics;    Visium spatial;    mouse brain transcriptome;    RNA quality;    RNA protection;    immunofluorescent staining;    methanol fixation;   

DOI  :  10.3389/fnins.2023.1198154

 received in 2023-03-31, accepted in 2023-05-03,  发布年份 2023

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

Unknown   
Copyright © 2023 Hahn, Bens, Kempfer, Reißig, Schmidl and Geis.