Single-cell kinetics of siRNA-mediated mRNA degradation

Krzyszton, Rafal; Woschee, Daniel; Reiser, Anita; Schwake, Gerlinde; Strey, Helmut H. und Rädler, Joachim O. (2019): Single-cell kinetics of siRNA-mediated mRNA degradation. In: Nanomedicine-Nanotechnology Biology and Medicine, Bd. 21, 102077

Abstract

RNA interference (RNAi) enables the therapeutic use of small interfering RNAs (siRNAs) to silence disease-related genes. The efficiency of silencing is commonly assessed by measuring expression levels of the target protein at a given time point post-transfection. Here, we determine the siRNA-induced fold change in mRNA degradation kinetics from single-cell fluorescence time-courses obtained using live-cell imaging on single-cell arrays (LISCA). After simultaneous transfection of mRNAs encoding eGFP (target) and CayRFP (reference), the eGFP expression is silenced by siRNA. The single-cell time-courses are fitted using a mathematical model of gene expression. Analysis yields best estimates of related kinetic rate constants, including mRNA degradation constants. We determine the siRNA-induced changes in kinetic rates and their correlations between target and reference protein expression. Assessment of mRNA degradation constants using single-cell time-lapse imaging is fast (<30 h) and returns an accurate, time-independent measure of siRNA-induced silencing, thus allowing the exact evaluation of siRNA therapeutics. (C) 2019 The Authors. Published by Elsevier Inc.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Physik
• Fakultätsübergreifende Einrichtungen: Center for NanoScience (CENS)
• Themengebiete: 500 Naturwissenschaften und Mathematik > 530 Physik ; 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
• ISSN: 1549-9634
• Sprache: Englisch