Optimization of Brain Tissue Preservation for Nucleic Acid Stability.

Area of Science:

• Neuroscience
• Molecular Biology
• Biotechnology

Background:

• Postmortem human brain tissue is vital for research.
• Spatial transcriptomics enables gene expression mapping within tissue context.
• Current methods using formalin-fixed, paraffin-embedded (FFPE) or frozen tissues present trade-offs between cytoarchitecture and RNA quality.

Purpose of the Study:

• To evaluate and optimize protocols for preparing postmortem human brain tissue for spatial transcriptomics.
• To assess the impact of postmortem interval and fixation methods on tissue integrity and RNA quality.

Main Methods:

• Sheep brains (n=16) were used to simulate human postmortem tissue preparation.
• RNA quality was assessed using RNA integrity number equivalent (RINe) for frozen tissue and DV200 for FFPE tissue.
• Cytoarchitectural integrity of cryosections was evaluated by quantifying tissue voids.

Main Results:

• Postmortem interval significantly decreased RINe in frozen tissue (7.2 at 24 hr to 4.8 at 168 hr).
• FFPE tissue DV200 values were highest (up to 69.01%) in samples fixed <24 hr.
• Liquid nitrogen pretreatment improved cryosection integrity (11.6% voids), while cryoprotectants had minimal impact.

Conclusions:

• Findings offer guidelines for optimizing postmortem tissue preparation for spatial transcriptomics.
• Freezing protocols need further development to match FFPE tissue's cytoarchitectural preservation for spatial transcriptomics applications.