1. Absolute quantification of noncoding RNA by microscale thermophoresis
- Author
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Raffael Schaffrath, Yuri Motorin, Kathrin Thüring, Mark Helm, Michael Stock, Roland Klassen, Akif Ciftci, Virginie Marchand, Sebastian A. Leidel, Jean-Yves Roignant, Aurellia Galliot, Adeline Galvanin, Karin Scharmann, Dominik Jacob, Johannes Gutenberg - Universität Mainz (JGU), Ingénierie, Biologie et Santé en Lorraine (IBSLor), Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), University of Freiburg [Freiburg], Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Institute of Molecular Biology (IMB), and Universität Kassel [Kassel]
- Subjects
tRNA stability ,RNA, Untranslated ,Absolute quantification ,RNA Quantification | Hot Paper ,Computational biology ,010402 general chemistry ,01 natural sciences ,Catalysis ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,RNA modification ,540 Chemistry ,hybridization ,ComputingMilieux_MISCELLANEOUS ,010405 organic chemistry ,Chemistry ,Microscale thermophoresis ,Communication ,RNA ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,General Chemistry ,Ribosomal RNA ,Non-coding RNA ,microscale thermophoresis ,Communications ,0104 chemical sciences ,Tissue Differentiation ,Transfer RNA ,570 Life sciences ,biology ,fluorescence ,RNA quantification - Abstract
Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA‐Seq‐based quantification approaches, strongly suggesting a bias due to tRNA modifications in the latter. Further applications include the quantification of rRNA as well as of polyA levels in cellular RNA.
- Published
- 2019