Sarin, L. P. | bioRxiv Science

bioRxiv2025

Rapid tRNA Isolation 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 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

molecular biology↗

bioRxiv2025

An efficient one-step rRNA depletion method for RNA sequencing in non-model organisms

RNA sequencing (RNA-seq) has revolutionized global transcriptomic analysis, ribosome footprinting, and polysome profiling, providing a wealth of data. Importantly, many RNA-based omics approaches typically involve either the removal of ribosomal RNA (rRNA) or selection of messenger RNA (mRNA) prior to sequencing, thereby enriching reads that map to the translationally active part of the transcriptome. Prokaryotic mRNA differs from eukaryotic mRNA in that it lacks the 3 polyadenylated tail, which excludes the use of poly(A)-based selection methods. While commercial rRNA depletion products exist for a growing number of prokaryotes, their proprietary nature and potential inefficiency with non-model organisms are factors that may limit broad-scale application. To mitigate this issue, we designed DepStep, a consolidated workflow for one-step rRNA depletion using species-specific biotinylated antisense probes for selective hybridization and removal of the target rRNA molecules. As a proof-of-concept, RNA-seq libraries of the psychrophilic gram-negative bacterium Shewanella glacialimarina TZS-4T were prepared using both DepStep and a commercial rRNA depletion kit for gram-negative bacteria, to which DepStep was benchmarked. DepStep compares favorably to the commercial depletion kit; it efficiently removes >98.6% of the rRNA content, and a slight increase in total read counts aligning to the coding sequences (CDS) was observed. Importantly, DepSteps cost-per-sample is three times lower than the commercial kit, establishing DepStep as a simple yet cost-effective alternative to commercial solutions.

molecular biology↗