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.
Item Type: | Journal article |
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Faculties: | Physics |
Research Centers: | Center for NanoScience (CENS) |
Subjects: | 500 Science > 530 Physics 500 Science > 500 Science |
ISSN: | 1549-9634 |
Language: | English |
Item ID: | 82470 |
Date Deposited: | 15. Dec 2021, 15:01 |
Last Modified: | 08. Nov 2023, 18:33 |