Protocol for rapid tRNA enrichment and chemiluminescent northern blot detection of tRNA and tRNA-derived fragments

Transfer RNA (tRNA), its post-transcriptional modifications, and tRNA-derived fragments (tRFs) play essential roles in cellular processes and gene regulation. Here, we present a fast and efficient tRNA enrichment protocol using silica spin columns. To analyze tRNA and tRFs, we describe steps for DNA probe labeling, tRNA separation on polyacrylamide (PAA) gels, and RNA transfer, UV crosslinking, and non-radioactive northern blotting. Additionally, this protocol describes the use of chemical affinity modifiers, enabling the detection of chemical modifications in specific tRNA isoacceptors. • Steps for tRNA enrichment from total RNA using silica spin columns • Procedure for DNA probe biotinylation via terminal deoxynucleotidyl transferase • Instructions for non-radioactive chemiluminescent northern blotting Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Transfer RNA (tRNA), its post-transcriptional modifications, and tRNA-derived fragments (tRFs) play essential roles in cellular processes and gene regulation. Here, we present a fast and efficient tRNA enrichment protocol using silica spin columns. To analyze tRNA and tRFs, we describe steps for DNA probe labeling, tRNA separation on polyacrylamide (PAA) gels, and RNA transfer, UV crosslinking, and non-radioactive northern blotting. Additionally, this protocol describes the use of chemical affinity modifiers, enabling the detection of chemical modifications in specific tRNA isoacceptors.