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Nanomedicine. 2019 Oct;21:102077. doi: 10.1016/j.nano.2019.102077. Epub 2019 Aug 8.

Single-cell kinetics of siRNA-mediated mRNA degradation.

Author information

1
Faculty of Physics, Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany; Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany; Nano systems Initiative Munich (NIM) and Center for NanoScience (CeNS), Munich, Germany. Electronic address: r.krzyszton@physik.lmu.de.
2
Faculty of Physics, Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany; Nano systems Initiative Munich (NIM) and Center for NanoScience (CeNS), Munich, Germany.
3
Faculty of Physics, Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany; Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany; Nano systems Initiative Munich (NIM) and Center for NanoScience (CeNS), Munich, Germany.
4
Faculty of Physics, Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany.
5
Department of Biomedical Engineering and Laufer Center for Quantitative Biology, Stony Brook University, Stony Brook, NY.
6
Faculty of Physics, Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany; Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-Universität Munich (LMU), Munich, Germany; Nano systems Initiative Munich (NIM) and Center for NanoScience (CeNS), Munich, Germany. Electronic address: raedler@lmu.de.

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.

KEYWORDS:

RNA interference; RNAi; kinetics; knockdown; siRNA; silencing

PMID:
31400572
DOI:
10.1016/j.nano.2019.102077
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