Enhanced RNA Preservation in Mouse Brain Tissue: A Strategy Combining Cardiac Perfusion with Hypersaline Immersion

One of the key research
methods for low-density spatial transcriptomics
involves collecting and sequencing microregions of interest from tissue
sections. However, prolonged capillary microneedle sampling poses
a risk of RNA degradation. To address this issue, we developed a preservation
strategy consisting of: (1) mouse cardiac perfusion with phosphate-buffered
saline (PBS) to reduce blood-borne RNase activity; (2) brain tissue
immersion in a hypersaline solution prior to sectioning; and (3) microregion
sampling under low-temperature conditions. Assessment of tissue section
RNA integrity and derived microregion transcriptomes demonstrated
that this combined approach effectively preserves RNA integrity, maintaining
an RNA integrity number equivalent greater than 7.5 for 6 h at 25
°C and greater than 8.5 for 24 h at 4 °C. Furthermore, this
protective method reduced 3′ transcript bias and enhanced the
detection of cell-type marker genes compared to controls (p < 0.05). This strategy establishes a standardized framework
to minimize preanalytical variability in capillary-based spatial omics
studies of mouse brain sections.