Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h
Sophie Seiffer1, Jana Brendler1, Angela Schulz2
1Institute of Anatomy, Medical Faculty, Leipzig University, Leipzig, Germany.
Abstract:
Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.
Insights
Post-mortem (PM) brain tissue allows RNA localization, including microglia-specific receptor RNA, within 24 hours of sampling. Storing cadavers at 4°C extends RNA detection, preserving spatial and cellular RNA localization.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Protein localization in post-mortem (PM) brain tissue is feasible with short post-mortem delays.
- RNA localization in PM brain tissue has not been extensively studied, particularly for specific cell types like microglia.
Purpose of the Study:
- To investigate the feasibility of RNA localization in PM mouse brain tissue using RNAscope Multiplex Fluorescent Detection.
- To assess the impact of post-mortem delays and storage conditions on RNA integrity and detection, focusing on microglia-specific RNAs.
Main Methods:
- Brains from mice with varying PM delays (up to 48 hours) and storage conditions (room temperature vs. 4°C) were processed into paraffin sections.
- RNAscope Multiplex Fluorescent Detection Kit v2 was used to detect ubiquitously expressed RNAs (Polr2a, PPIB, UBC) and microglia-specific receptor RNAs (Gpr34, P2ry12).
- Simultaneous labeling with microglia-specific antibodies (Iba1 or P2ry12) enabled cellular co-localization of detected RNAs.
Main Results:
- Ubiquitous RNAs were reliably detected up to 48 hours PM at room temperature.
- Microglia-specific receptor RNAs (Gpr34, P2ry12) were successfully localized to microglia within 24 hours PM.
- Significant reduction in receptor RNA signals occurred after 48 hours PM at room temperature, but this was mitigated by storage at 4°C.
Conclusions:
- Spatial and cellular localization of specific RNA is achievable in PM brain tissue sections within 24 hours of sampling.
- RNA integrity for detection is better preserved at 4°C compared to room temperature for longer PM delays.
- This method enables the study of RNA expression in microglia and other cell types in PM brain samples.
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