Rapid tRNA Isolation and Chemiluminescent Northern Blot Detection of tRNA and tRNA-Derived Fragments

Abstract

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 isolation using silica spin columns. To analyze the isolated tRNA and detect tRFs, we describe a sensitive and cost-effective non-radioactive Northern blotting technique. Additionally, this blotting method is compatible with chemical affinity modifiers, such as [p-(N-acrylamino)-phenyl]mercuric chloride (APM) or 3-(acrylamido)phenylboronic acid (APB) enabling the detection of chemical modifications in specific tRNA isoacceptors. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/681074v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1b0618forg.highwire.dtl.DTLVardef@d507a4org.highwire.dtl.DTLVardef@1e8b6faorg.highwire.dtl.DTLVardef@1450326_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIRapid (<30 min) tRNA isolation from total RNA using silica containing spin columns C_LIO_LIProcedure for DNA probes biotinylation via terminal deoxynucleotidyl transferase C_LIO_LINon-radioactive chemiluminescent Northern blotting C_LI