Nucleic Acids Research, 2005, Vol. 33, No. 22 e190 doi:10.1093/nar/gni188 Quantum dots to monitor RNAi delivery and improve gene silencing Alice A. Chen 1 , Austin M. Derfus 2 , Salman R. Khetani1,2 and Sangeeta N. Bhatia1,2,3,* 1Harvard-M.I.T. Division of Health Sciences and Technology/Electrical Engineering and Computer Science, Massachusetts Institute of Technology, MA, USA, 2Department of Bioengineering, University of California at San Diego, CA, USA and 3Division of Medicine, Brigham & Women’s Hospital, Boston, MA, USA Received May 24, 2005; Revised and Accepted November 22, 2005 ABSTRACT A critical issue in using RNA interference for identify- ing genotype/phenotype correlations is the uniform- ity of gene silencing within a cell population. Variations in transfection efficiency, delivery- induced cytotoxicity and ‘off target’ effects at high siRNA concentrations can confound the interpreta- tion 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 nano- particles 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 plas- mids 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 sub- population of highly silenced cells obtained by QD labeling was required to observe significant down- stream effects of gene knockdown. INTRODUCTION RNA interference (RNAi) has emerged as a powerful tool for studying gene function. Since the discovery of RNAi (1), the evolutionarily conserved cellular process has been exploited to determine the functions of nearly every gene in model organisms Caenorhabditis elegans (2,3) and Drosophila melanogaster (4) and a host of mammalian genes including 23% of the sequenced human genes (5,6). If the RNAi effector sequence is potent and the siRNA delivered efﬁciently throughout the cell culture, remarkably speciﬁc post- transcriptional inhibition of gene expression can be achieved (7,8). However, inefﬁcient and heterogeneous delivery of siRNA is frequently observed in cell cultures, causing variable levels of gene silencing and potentially confounding the inter- pretation of genotype/phenotype correlations (9–12). Without the means to address and resolve transfection variability, the utility of RNAi in eukaryotes will be limited only to mam- malian cell types that have been thoroughly optimized for siRNA delivery (13). The importance of high transfection efﬁciency has been spotlighted among numerous reports investigating methods to either improve RNAi delivery (14–17) or screen for efﬁcient knockdown. In the latter case, typical strategies involve monitoring ﬂuorescently end-modiﬁed siRNAs (18,19) or co-transfecting reporter plasmids and selecting for high trans- fection by ﬂuorescence or antibiotic-resistance (20). These techniques enable one-time selection of highly transfected cells yet discard moderately-silenced cells, which may be of interest to the study. For example, varying degrees of RNAi-mediated down-regulation in the tumor suppressor gene Trp53 have been shown to modulate expression of dis- tinct pathological phenotypes both in vitro and in vivo (21). Moreover, rapid photobleaching of organic ﬂuorophores and *To whom correspondence should be addressed at Laboratory for Multiscale Regenerative Technologies, 77 Massachusetts Avenue, E19-502D, Cambridge, MA 02139, USA. Tel: +617 324 0221; Fax: +617 324 0740; Email: sbhatia@mit.edu The authors wish it be known that, in their opinion, the first two authors should be regarded as joint First Authors The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org