Molecular Analysis of miRNA Stability and Decay Dynamics by Northern Blot.

MicroRNAs (miRNAs) are critical post-transcriptional regulators that function through association with Argonaute proteins to form the RNA-induced silencing complex (RISC). Although miRNAs were considered remarkably stable molecules, their cellular abundance is subject to active regulation through coordinated degradation pathways. Precise quantification of miRNA stability remains technically challenging, primarily owing to the small size of these molecules, their elevated GC content, and their relatively low abundance compared to other cellular RNA species. To overcome these technical limitations, this chapter provides a comprehensive experimental strategy for monitoring miRNA stability using optimized northern blotting techniques. This approach integrates transcriptional shutoff through actinomycin D treatment with advanced detection chemistries-specifically, EDC-mediated crosslinking and near-infrared fluorescent probe hybridization-to markedly enhance signal resolution and detection sensitivity, thereby facilitating precise determination of decay kinetics and reliable calculation of miRNA half-lives. Collectively, this method offers high specificity, enhanced sensitivity, and quantitative accuracy to investigate miRNA degradation dynamics at single-species resolution.