Calcified Artery Preparation and Processing with Preserved Morphology and RNA for Digital Spatial Profiling

Area of Science:

• Vascular Biology
• Spatial Transcriptomics
• Genomics

Background:

• Spatial transcriptomics revolutionized many biological fields but is underutilized in vascular biology.
• Vascular diseases are a leading cause of death, necessitating advanced research tools.
• Challenges in preparing vascular tissues, especially diseased ones, hinder spatial transcriptomics adoption.

Purpose of the Study:

• To develop and present a detailed, step-by-step protocol for spatial transcriptomics in human tibial arteries.
• To address challenges in tissue handling, fixation, decalcification, and RNA integrity for vascular samples.
• To enable layer-specific transcriptional analysis in healthy and diseased vessels.

Main Methods:

• Optimized workflow for human tibial artery tissue preparation, including fixation and decalcification.
• Histological staining for calcification severity grading.
• Tissue microarray (TMA) construction and region of interest (ROI) selection on NanoString GeoMx Digital Spatial Profiler (DSP).

Main Results:

• A protocol that preserves vascular tissue morphology and RNA quality during processing.
• Successful application of spatial transcriptomics to complex vascular specimens.
• Demonstration of layer-specific transcriptional activity analysis in arterial walls.

Conclusions:

• The developed protocol lowers technical barriers for spatial transcriptomics in vascular research.
• This method facilitates the study of vascular pathologies like atherosclerosis and calcification.
• The protocol is expected to accelerate discoveries in vascular biology and promote wider technique adoption.