ribogreen rna fluorescence assay  (Thermo Fisher)

Journal: bioRxiv

Article Title: Modular mRNA lipid nanoparticle platform rescues diverse genetic male infertility

doi: 10.1101/2025.11.13.688269

Figure Lengend Snippet: (a) Upper: Schematic of the Ai9 Cre-reporter system. In Ai9 mice, a LoxP-flanked STOP cassette prevents tdTomato expression until Cre recombinase excises the STOP, activating red fluorescence in any cell that has received Cre. Lower: In vivo fluorescence imaging of Ai9 mice 48 h after intratubular injection of Cre mRNA LNP (n = 3 mice per group). H, heart; Lu, lung; Li, liver; S, spleen; K, kidney; T, testis. (b) A seminiferous tubule cross-section from a treated testis, co-stained for tdTomato (red), the Sertoli cell marker SOX9 (green), and nuclei (DAPI, blue). Scale bar: 40 µm. (c) Upper: Gating strategy to resolve stages: pre-leptotene spermatogonia (2N, within P5), leptotene/zygotene spermatocytes (replicating DNA, P6), pachytene spermatocytes (4N, mid-meiosis, P7), diplotene spermatocytes (4N, late meiosis, P8), secondary spermatocytes (2N, post-meiosis II, P9) and round spermatids (1N, P10). Lower: Percentage of tdTomato-positive cells within each gated population for ALC-0315, MC3 versus SM-102 LNP–Cre-treated testes (48 h post-injection; n = 6 testes each). (d) Kinetics of eGFP expression in testes following delivery of different RNA formats. Mice were injected (via MC3 LNP) with either N¹mΨ-modified eGFP mRNA (red), self-amplifying RNA replicon (saRNA, black) or circular RNA (circRNA, blue) encoding eGFP. Plotted is the radiant efficiency (GFP fluorescence) of the injected testis over 14 days (mean ± s.d.; n = 3 mice per group). (e) Time-course comparison of unmodified versus pseudouridine-modified mRNA (both encoding eGFP). Unmodified mRNA (black) drives an early peak of GFP (within 1 day) followed by a steep decline; modified mRNA (red) shows a delayed peak (∼3 days) and a slower decline (mean ± s.d.; n = 3 mice per group). (f) RT-qPCR measurement of eGFP mRNA in the testis at various times after injection of unmodified versus modified eGFP mRNA. (g) Innate immune gene induction in the testis by unmodified versus modified mRNA. RT-qPCR measurements were taken at 6, 12 and 24 h after injection of 10 µg eGFP mRNA (either unmodified or N¹mΨ-modified). Data are mean ± s.d. (n = 3 mice per group).

Article Snippet: Encapsulation efficiency of mRNA was assessed with a RiboGreen RNA fluorescence assay (Thermo Fisher Quant-iTTM RiboGreen kit), by treating aliquots of the LNP with or without 0.2% Triton X-100 to distinguish encapsulated vs. total RNA.

Techniques: Expressing, Fluorescence, In Vivo, Imaging, Injection, Staining, Marker, Modification, Comparison, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Determining the effects of pseudouridine incorporation on human tRNAs

doi: 10.1038/s44318-025-00443-y

Figure Lengend Snippet: ( A ) Top: Scheme of in vitro transcription (IVT) production of human cytosolic tRNAs. DNA templates contain T7 polymerase recognition site and tRNA sequence with 5ʹ GG. Bottom: Urea gel showing purity of produced tRNAs. ( B ) nanoDLS determined hydrodynamic radius (rH) profiles of each in vitro transcribed tRNAs. 2.5 nm corresponds to rH of folded tRNA, 3.1 nm corresponds to rH of partially unfolded tRNA, 5.0 nm corresponds to rH of unfolded tRNA. Shaded regions around the curves represent s.d. error. Biological replicates, N = 3. ( C ) Plot of experimentally determined melting temperatures (Exp Tm, °C) of in vitro transcribed tRNAs determined using Quant-iT RiboGreen™ RNA fluorescent probe versus GC content (%). The dashed line corresponds to 37 °C. tRNA Val UAC and tRNA Pro UGG have no inflection point in Tm determination. ( D ) Representative micrograph and 2D class averages of human tRNA Gln UUG (upper left) and tRNA Gly CCC (lower left). Cryo-EM reconstructions of human tRNA Gln UUG (upper middle) and tRNA Gly CCC (lower middle) with an ensemble of 10 atomic models fit into the density (right). Inset: Clover-leaf secondary structure representation of tRNA highlighted with each domain: acceptor arm (AAS, green); T-arm (yellow) with highlighted C 56 (dark yellow); D-arm (blue) with highlighted G 19 (dark blue); variable loop (VL, gray); anticodon arm (ASL, pink). 5ʹ and 3ʹ ends are indicated. All cryo-EM maps are contoured to RMSD = 8. .

Article Snippet: The thermal stability of the tRNAs was evaluated with the use of a Quant-iT RiboGreenTM RNA fluorescent probe (RiboGreen) ( R11491 ThermoFisher Scientific).

Techniques: In Vitro, Sequencing, Produced, Cryo-EM Sample Prep