Detection of tRNA-Derived Small RNAs in Arabidopsis thaliana via Northern Blotting: Exploring Potential Biomarkers for Phytohormone Analysis.

Noncoding small RNAs (sRNAs) are well-known regulatory molecules that influence a wide range of cellular processes in eukaryotes and often function in concert with phytohormones. Advances in next-generation sequencing (NGS) have further expanded the identification and characterization of sRNAs from diverse sources, highlighting their potential as early biomarkers for hormone and stress signaling. In recent work, we identified transfer RNA-derived fragments (tRFs) as a prominent class of sRNAs, induced as early as 1 h post-infection with Pseudomonas syringae, that positively regulates defense gene expression in Arabidopsis. However, verifying these sRNA findings from NGS data presents a technical challenge, as the small size and often low abundance of these sRNAs complicate accurate detection and quantification. Here, we present a procedure to enrich sRNAs and facilitate the detection of sRNAs in Arabidopsis using a sensitive northern blot protocol. We evaluated sRNA enrichment using the conventional Trizol method with and without nucleic acid precipitants, as well as commercial column-based extraction kits. Our northern blot results show that total RNA purified by both methods is enriched with sRNAs. Additionally, the precipitants, such as linear acrylamide and glycogen, improved the detection of tRFs. Column-based kits also yielded comparable enrichment of tRFs and provided a rapid and straightforward protocol essential for preserving RNA integrity. This study thus presents a robust, nonradioactive and PCR-free detection method for characterizing the regulatory functions of sRNAs.