Complementary Approaches for Accurate Detection of tRNA-Derived Small RNAs (tsRNAs): Small RNA Northern Blotting and...

tRNA-derived small RNAs (tsRNAs) are generated by specific cleavage of transfer RNAs that are essential for delivering amino acids during protein synthesis. They have emerged as important regulators of gene expression across diverse biological contexts and have been implicated in multiple diseases, including cancers and neurological disorders. Advances in next-generation sequencing (NGS) have enabled high-throughput profiling of tsRNA abundance; however, extensive base modifications and heterogeneous termini frequently lead to inaccurate outcomes in small RNA sequencing. These technical limitations highlight the need for reliable validation strategies. Here, we present two complementary methods for tsRNA validation: small RNA northern blotting, which allows direct visualization of RNA size and abundance, and dumbbell PCR, which enables isoform-specific quantification with single-nucleotide resolution. Together, these approaches provide robust tools to validate tsRNA expression and facilitate mechanistic studies of their biological functions.