Single-Cell Kinetics of siRNA-Mediated mRNA Degradation

Publication date: Available online 8 August 2019Source: Nanomedicine: Nanotechnology, Biology and MedicineAuthor(s): Rafał Krzysztoń, Daniel Woschée, Anita Reiser, Gerlinde Schwake, Helmut H. Strey, Joachim O. RädlerAbstractRNA 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.Graphical AbstractUsing Live-cell imaging on single-cell arrays (LISCA) we monitor the fluorescence intensity time-courses of reporter protei...
Source: Nanomedicine: Nanotechnology, Biology and Medicine - Category: Nanotechnology Source Type: research