Detecting RNA base methylations in single cells by in situ hybridization

Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~10(4)–10(7) cells are used to detect these modifications, obscuring potent...

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Detalles Bibliográficos
Autores principales: Ranasinghe, Rohan T., Challand, Martin R., Ganzinger, Kristina A., Lewis, Benjamin W., Softley, Charlotte, Schmied, Wolfgang H., Horrocks, Mathew H., Shivji, Nadia, Chin, Jason W., Spencer, James, Klenerman, David
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811446/
https://www.ncbi.nlm.nih.gov/pubmed/29440632
http://dx.doi.org/10.1038/s41467-017-02714-7
Descripción
Sumario:Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~10(4)–10(7) cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m(6)(2)A, m(1)G and m(3)U) that destabilize Watson–Crick base pairs. Our method—methylation-sensitive RNA fluorescence in situ hybridization—detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging.

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