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Angew Chem Int Ed Engl. 2019 Mar 20. doi: 10.1002/anie.201814377. [Epub ahead of print]

Absolute quantification of noncoding RNA by microscale thermophoresis.

Author information

1
Johannes Gutenberg Universitat Mainz, Institut für Pharmazie und Biochemie, Staudingerweg 5, 55128, Mainz, GERMANY.
2
Universite de Lorraine, biopole UL, 9, Avenue de la Forêt de Haye, 54505, Vandoeuvre-les-Nancy, FRANCE.
3
Albert-Ludwigs-Universitat Freiburg, Institut für Biochemie und Molekularbiologie, Stefan-Meier-Straße 17, 79104, Freiburg, GERMANY.
4
Max-Planck-Institut fur molekulare Biomedizin, RNA biology, Von-Esmarch-Strasse 54, 48149, Münster, GERMANY.
5
Institute of Molecular Biology Mainz, Ackermannweg 4, Mainz, 55128, GERMANY.
6
Universite de Lorraine, Biopole Ul, 9, Avenue de la Forêt de Haye, 54505, Vandoeuvre-les-Nancy, FRANCE.
7
Universitat Kassel, Institut für Biologie, Mikrobiologie, Heinrich-Plett-Str. 40, 34132, Kassel, GERMANY.
8
Johannes-Gutenberg-Universität Mainz, Institut für Pharmazie und Biochemie, Staudinger Weg 5, Not Available, 55128, Mainz, GERMANY.

Abstract

Accurate quantification of 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. Here we present a hybridization-based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify e.g. single tRNA species with a turnaround time of about one hour. We developed MST in to quantify the effect of tRNA toxins and of heat stress and RNA modification. 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 included quantification of rRNA, as well as of polyA levels in cellular RNA.

KEYWORDS:

Fluorescence; Microscale Thermophoresis; RNA quantification; hybridization; tRNA stability

PMID:
30892798
DOI:
10.1002/anie.201814377

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