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.