Rolling circle amplification for next-generation molecular diagnostics, genome analysis, and spatial transcriptome profiling

RNA sequencing and hybridization have enabled high-resolution gene expression mapping in tissue architecture, providing invaluable insights into cellular heterogeneity and disease microenvironments. This review comprehensively explores the fundamental principles, design modifications, and advanced signal amplification strategies of RCA, with a focus on its applications in molecular diagnostics, genome analysis, and spatial transcriptome profiling. Additionally, it addresses key challenges, including reaction specificity, automation, and cost-effectiveness, and discusses future directions for integrating RCA into high-throughput molecular diagnostics, biomarker discovery, and single-cell and spatial multi-omics platforms. Through continuous innovation, RCA is poised to play a transformative role in precision medicine, next-generation sequencing, and biomedical research.