Approximately 100 mg of whole-brain tissue was collected from 14.5 dpc embryos and homogenized with ceramic beads (Sapphire Bioscience) using TRIzol reagent (Life Technologies) in tissue homogenizer (Bertin Technologies). Total RNA and tRNA isolation, tRNA hydrolysis, and tRNA modification analysis via high-performance liquid chromatography coupled to mass spectrometry was performed as previously described26 (link). Mass spectrometer parameters were optimized for targeted ribonucleosides using multiple injections of 0.1–1 ng of commercially obtained m7G (MetaGene) and purified ncm5U, mcm5U, and mcm5s2U nucleosides (a generous gift from Sebastian Leidel, University of Bern, Switzerland). The obtained retention times were superimposed with the previously published results94 (link). An analytical method was developed for the simultaneous analysis of modified ribonucleosides of which six compounds of interest were targeted for quantification (ncm5U, mcm5U, mcm5s2U, Ψ, m1A, m7G, and t6A). Pseudouridine (Ψ) served as an internal normalization standard. MultiQuant v2.1.1 (AB Sciex) software was used for peak assignment, quantification, and normalization.
Kojic M., Gawda T., Gaik M., Begg A., Salerno-Kochan A., Kurniawan N.D., Jones A., Drożdżyk K., Kościelniak A., Chramiec-Głąbik A., Hediyeh-Zadeh S., Kasherman M., Shim W.J., Sinniah E., Genovesi L.A., Abrahamsen R.K., Fenger C.D., Madsen C.G., Cohen J.S., Fatemi A., Stark Z., Lunke S., Lee J., Hansen J.K., Boxill M.F., Keren B., Marey I., Saenz M.S., Brown K., Alexander S.A., Mureev S., Batzilla A., Davis M.J., Piper M., Bodén M., Burne T.H., Palpant N.J., Møller R.S., Glatt S, & Wainwright B.J. (2021). Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype. Nature Communications, 12, 2678.
Other organizations :
Translational Research Institute, University of Queensland, Jagiellonian University, Walter and Eliza Hall Institute of Medical Research, Hvidovre Hospital, Kennedy Krieger Institute, University of Melbourne, Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Sorbonne Université, Assistance Publique – Hôpitaux de Paris, Pitié-Salpêtrière Hospital, University of Colorado Denver, Children's Hospital Colorado, ARC Centre of Excellence in Synthetic Biology, Commonwealth Scientific and Industrial Research Organisation, Queensland University of Technology
Approximately 100 mg of whole-brain tissue collected from 14.5 dpc embryos
dependent variables
TRNA modification analysis via high-performance liquid chromatography coupled to mass spectrometry
Quantification of six targeted modified ribonucleosides: ncm5U, mcm5U, mcm5s2U, Ψ, m1A, m7G, and t6A
control variables
Ceramic beads (Sapphire Bioscience) used for homogenization
TRIzol reagent (Life Technologies) used for total RNA and tRNA isolation
Tissue homogenizer (Bertin Technologies) used for homogenization
Commercially obtained m7G (MetaGene) and purified ncm5U, mcm5U, and mcm5s2U nucleosides (a generous gift from Sebastian Leidel, University of Bern, Switzerland) used for mass spectrometer parameter optimization
Pseudouridine (Ψ) used as an internal normalization standard
MultiQuant v2.1.1 (AB Sciex) software used for peak assignment, quantification, and normalization
Annotations
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