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- W2079268506 abstract "RNA interference (RNAi) is a gene regulation pathway induced by small RNA molecules, microRNA (miRNA) and small interfering RNA (siRNA). It is a multi-step process consisting of the small RNA genesis by Dicer, guide strand selection and the guide strand-mediated gene silencing either through mRNA cleavage or translation inhibition by RNA-induced silencing complex (RISC). Even though RNAi is one of the major techniques to regulate gene, the mechanistic details of each step are yet to be determined. Especially, unveiling which step is the rate-determining step would deepen the understanding about the molecular mechanism of RNAi and could contribute designing more efficient small RNAs for gene silencing. To address the question, the quantitative analysis of RNAi is required, which benefits from the detection of single mRNA at single-cell level. Here, we incorporated single-molecule fluorescence in situ hybridization (smFISH) to count mRNA at single-cell level and screened multiple sets of small RNAs which mimic miRNA, siRNA, pre-miRNA and pre-siRNA. Together with single-molecule fluorescence resonance energy transfer (smFRET) which enables us to quantify the RNA cleavage by Dicer, we discovered that the cleavage step by Dicer controls the overall silencing kinetics. Interestingly, we also found that Dicer is sensitive to 3’ overhang not RISC. The quantitative analysis of RNAi using smFISH together with smFRET would be a powerful platform to study RNAi and other gene-regulating system." @default.
- W2079268506 created "2016-06-24" @default.
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- W2079268506 date "2015-01-01" @default.
- W2079268506 modified "2023-09-29" @default.
- W2079268506 title "Quantitative Analysis of RNA Interference by mRNA Couting at Single-Cell Level" @default.
- W2079268506 doi "https://doi.org/10.1016/j.bpj.2014.11.1998" @default.
- W2079268506 hasPublicationYear "2015" @default.
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