Impact of fixation duration on messenger RNA detectability in human formalin-fixed paraffin-embedded brain tissue
Charlene-Annett Hurler1, Sabine Liebscher2,3,4,5, Thomas Arzberger6,7
1Institute for Stroke and Dementia Research, LMU University Hospital, Ludwig-Maximilians-University Munich, 81377 Munich, Germany.
Abstract:
Technologies to study mRNA in post-mortem human brain samples have greatly advanced our understanding of brain pathologies. With ongoing improvements, particularly in formalin-fixed paraffin-embedded tissue, these technologies will continue to enhance our knowledge in the future. Despite various considerations for tissue and mRNA quality, such as pre-mortem health status and RNA integrity, the impact of the tissue fixation time has not been addressed in a systemic fashion yet. In this study, we employed RNAscope to assess mRNA detectability in human post-mortem brain tissue in relation to fixation time. Our results reveal a dynamic change in mRNA detection across varying fixation durations, accompanied by an increase in signal derived from the negative probe and autofluorescence background. These findings highlight the critical relevance of standardized fixation protocols for the collection of human brain tissue in order to probe mRNA abundancy to ensure reliable and comparable results.
Insights
Fixation time critically impacts mRNA detection in post-mortem human brain tissue. Standardizing fixation protocols is essential for reliable mRNA abundance studies in neuropathology research.
Area of Science:
- Neuroscience
- Molecular Biology
- Pathology
Background:
- mRNA analysis in post-mortem brain tissue is crucial for understanding brain pathologies.
- Formalin-fixed paraffin-embedded (FFPE) tissue technologies are advancing, but standardization is needed.
- The effect of tissue fixation duration on mRNA detectability remains understudied.
Purpose of the Study:
- To investigate the impact of fixation time on mRNA detectability in human post-mortem brain tissue.
- To assess changes in mRNA signal and background noise with varying fixation durations.
- To emphasize the need for standardized fixation protocols in neuropathology.
Main Methods:
- Utilized RNAscope technology for mRNA detection.
- Analyzed human post-mortem brain tissue samples with different fixation times.
- Quantified mRNA signal, negative probe signal, and autofluorescence.
Main Results:
- mRNA detectability showed dynamic changes across different fixation durations.
- An increase in signal from the negative probe was observed with longer fixation times.
- Autofluorescence background also increased with extended fixation periods.
Conclusions:
- Fixation time significantly influences mRNA detectability and signal integrity in post-mortem brain tissue.
- Standardized tissue fixation protocols are critical for ensuring reliable and comparable mRNA abundance data.
- These findings underscore the importance of optimizing fixation procedures for neuropathological research.
