Single-cell kinetics of siRNA-mediated mRNA degradation.
Nanomedicine
; 21: 102077, 2019 10.
Article
em En
| MEDLINE
| ID: mdl-31400572
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.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
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Transfecção
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Estabilidade de RNA
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RNA Interferente Pequeno
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Proteínas de Fluorescência Verde
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Ciências Biocomportamentais
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article