Sniffing for Gene-Silencing Efficiency of siRNAs in HeLa Cells in Comparison with That in HEK293T Cells: Correlation Between Knockdown Efficiency and Sustainability of siRNAs Revealed by FRET-Based Probing
| Autor: | Jisu Kim, Sang Soo Hah, Seonmi Shin, Yea Seul Kim, Dong-Eun Kim, Hyun-Mi Kwon |
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| Rok vydání: | 2013 |
| Předmět: |
Small interfering RNA
Transfection Biochemistry HeLa Original Research Articles Drug Discovery Fluorescence Resonance Energy Transfer Genetics Gene Knockdown Techniques Humans Nanotechnology Gene silencing Gene Silencing RNA Small Interfering Molecular Biology Fluorescent Dyes Gene knockdown biology HEK 293 cells biology.organism_classification Cell biology HEK293 Cells Förster resonance energy transfer Molecular Medicine HeLa Cells |
| Zdroj: | Nucleic Acid Therapeutics. 23:152-159 |
| ISSN: | 2159-3345 2159-3337 |
| DOI: | 10.1089/nat.2012.0396 |
| Popis: | Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules. |
| Databáze: | OpenAIRE |
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