Quantum dots to monitor RNAi delivery and improve gene silencing.

Publication Type:

Journal Article

Source:

Nucleic Acids Res, Volume 33, Issue 22, p.e190 (2005)

Keywords:

Animals, Cadherins, Coculture Techniques, Female, Flow Cytometry, Fluorescent Dyes, Quantum Dots, Rats, Rats, Inbred Lew, RNA Interference, RNA, Small Interfering, Transfection

Abstract:

<p>A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and &#39;off target&#39; effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell-cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown.</p>

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