Dynamics of gene silencing by RNA interference

Abstract

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.

Subcellular Fate and Off-Target Effects of siRNA, shRNA, and miRNA

“…These include mathematical model of RNAimediated gene silencing by Bergstrom and co-workers and Groenenboom and colleagues to understand mechanism of gene silencing due to transgene or virus (95,96). Raab and Stephanopoulos examined the dynamics of dose-and time dependent gene silencing by siRNA relative to plasmid transfection (97). Bartlett and Davis have combined intracellular imaging with mathematical modeling to trace siRNA induced RNAi from initial delivery to final intracellular function.…”

Moment analysis for kinetics of gene silencing by RNA interference

“…Time-dependent changes in RNAi effect and kinetic analysis of the effect have been reported in the literature (Hahn et al, 2004;Haley and Zamore, 2004;Raab and Stephanopoulos, 2004). To determine the optimal time point for mRNA analysis by qRT-PCR, Hahn et al (2004) studied the kinetics of mRNA degradation caused by siRNA.…”

“…Therefore, their study was limited to using cell extracts as the RNAi machinery and they did not study the induction of RNAi in living cells. The kinetics of transgene expression was discussed after the cotransfection of pDNA encoding reporter gene and the corresponding specific siRNA (Raab and Stephanopoulos, 2004). They attempted to predict the time-course of the transgene expression resulting from the transfection of pDNA and siRNA-based transcript degradation.…”

Applicability of Quantum Dots in Biomedical Science

“…Capping core nanocrystals with ZnS has shown to increase stability and performance, producing QDs with improved photophysical and chemical properties at room temperature. However, ZnS capping alone is not sufficient to stabilize the core, particularly in biological solutions [24]. Quantum dots do not exhibit aqueous solubility as they are generally synthesized in organic solution.…”