Journal: RNA Biology

Article Title: Production of MarathonRT and its comparison with commercial reverse transcriptases for tRNA sequencing library preparation

doi: 10.1080/15476286.2026.2720028

Figure Lengend Snippet: MRT and MRT-CBD construct design, purification, storability, and optimal enzyme-to-template ratio. (A) Schematic representation (not to scale) of MRT and updated MRT-CBD construct in a new plasmid backbone with a C-terminal CBD domain. (B) Overview of the optimized one-step protein production workflow. (C) Representative 10% SDS-PAGE gel (1.5 mm) of MRT and MRT-CBD expression. in – pooled elution fractions from affinity purification; conc – concentrated protein. Bands representing full-length protein are indicated (dot = MRT, triangle = MRT-CBD), colours correspond to panel A. Marker: PageRuler broad range unstained protein ladder. (D) Representative Western blot of concentrated protein detected by anti-His antibody. Full-length protein indicated by dot (MRT) or triangle (MRT-CBD). Densitometry analysis of the full-length protein is shown next to the plot, full-length protein band marked by blue dashed line. (E, F) Protein storability. Top panels: activity of samples stored at −20°C; bottom panels: activity of samples stored at −70°C. Measurements were performed in technical duplicates, except the first time point ( n = 3). Black outline around shapes indicates the use of a new RNA template batch. (E) protein activity over 52 weeks. (F) Activity during repeated freeze-thaw cycles over 20 weeks. (G) Bar plot of densitometry analysis of free cDNA produced by indicated enzyme amount (U) from 100 ng of 3’-adapter-ligated tRNA template. 0.5 units of the same MRT-CBD reaction were loaded on both gels for comparison.

Article Snippet: Bands corresponding to 5.8S and 5S rRNA are indicated. (B) binding capacity of Monarch RNA Cleanup columns (10 μg, NEB) loaded with 10, 5, or 2.5 μg total RNA.

Techniques: Construct, Purification, Plasmid Preparation, SDS Page, Expressing, Affinity Purification, Marker, Western Blot, Analysis, Activity Assay, Produced, Comparison

Journal: RNA Biology

Article Title: Production of MarathonRT and its comparison with commercial reverse transcriptases for tRNA sequencing library preparation

doi: 10.1080/15476286.2026.2720028

Figure Lengend Snippet: tRNA column and gel extraction for sequencing. (A) schematic overview of the tRNA silica-based spin column purification process. First, the total RNA sample is mixed with long RNA binding buffer (LBB) and bound to the first column. The flowthrough will contain RNAs < 120 bp. This flowthrough is mixed with short RNA binding buffer (SBB) and bound to the second column. The RNA bound to the column is then washed with a chaotropic wash (CW), ethanol wash (EW) and eluted with ultra-pure water. DA/DPH denotes deacylated and dephosphorylated total RNA and encircled roman numerals I and II correspond to eluates from column I and II, respectively. (B–D) RNA samples analysed on 8–10% denaturing urea-PAA gels. Marker: low‑range ssRNA (NEB). Bands corresponding to 5.8S and 5S rRNA are indicated. (B) binding capacity of Monarch RNA Cleanup columns (10 μg, NEB) loaded with 10, 5, or 2.5 µg total RNA. 50 ng RNA loaded to gel from column I and II eluates. input – total RNA post-phenol/BCP extraction; DA/DPH – deacylated and dephosphorylated total RNA; CTRL – only dephosphorylated total RNA; I/II – eluates from column I and II. (C) urea-PAA analysis of eluate from column I and II. Note removal of rRNA in eluate II. Equal volumes were loaded of both column eluates. (D) column enriched and gel-extracted tRNA used in the tRNA-seq library preparation. (E) PCA clustering of sequenced samples ( n = 48). Encircled areas correspond to column enriched tRNA (green ellipse) or gel-extracted tRNA (black ellipse). (F) correlation of normalized peak heights of global tRNA modifications detected by LC-MS between extraction methods (log 2 scale; column extraction n = 5, gel extraction n = 3). Pearson’s r = 0.9998, R 2 = 0.9995.

Article Snippet: Bands corresponding to 5.8S and 5S rRNA are indicated. (B) binding capacity of Monarch RNA Cleanup columns (10 μg, NEB) loaded with 10, 5, or 2.5 μg total RNA.

Techniques: Gel Extraction, Sequencing, Purification, RNA Binding Assay, Marker, Binding Assay, Extraction, Analysis, Liquid Chromatography with Mass Spectroscopy

Journal: RNA Biology

Article Title: Production of MarathonRT and its comparison with commercial reverse transcriptases for tRNA sequencing library preparation

doi: 10.1080/15476286.2026.2720028

Figure Lengend Snippet: Comparison of tRNA enrichment efficiency by extraction method.

Article Snippet: Bands corresponding to 5.8S and 5S rRNA are indicated. (B) binding capacity of Monarch RNA Cleanup columns (10 μg, NEB) loaded with 10, 5, or 2.5 μg total RNA.

Techniques: Comparison, Extraction, Concentration Assay, Gel Extraction