RNA Detection Technologies: A MethodCentric Guide to Principles and Reproducibility

RNA detection techniques have expanded into a diverse methodological landscape spanning hybridization, amplification, imaging, and sequencing. In this review, we provide a methodcentric synthesis of the major technologies that define this landscape, emphasizing how each method's core principle, practical strengths, and sources of variability shape its reproducibility. Beginning with foundational approaches, we trace the development of isothermal amplification, quantitative and digital PCR, microarrays, singlemolecule imaging, multiplexed spatial methods, and amplificationfree digital quantification. We then examine the transformative impact of bulk, singlecell, longread, directRNA, and spatial transcriptomics, as well as CRISPRbased detection and metabolic labeling for RNA dynamics. Across these technologies, we focus on reproducibility as a defining dimension of evaluation: mature methods benefit from established standards, whereas newer approaches remain prestandardization and require careful, experimentspecific controls. Rigorous method selection must be guided by the biological question, required resolution, sample constraints, and the maturity of each method's reproducibility framework. We conclude that RNA detection methods form interconnected methodological paths of problemsolving rather than simple replacements.