Quantum dots to monitor RNAi delivery and improve gene silencing.
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 'off target' 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.
Chen
Alice A
AA
Harvard-M.I.T. Division of Health Sciences and Technology/Electrical Engineering and Computer Science, Massachusetts Institute of Technology, MA, USA.
Derfus
Austin M
AM
Khetani
Salman R
SR
Bhatia
Sangeeta N
SN
D000818:Animals; D015820:Cadherins / Q000235:genetics; D018920:Coculture Techniques; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D045663:Quantum Dots; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000032:analysis*; D051381:Rats; D011917:Rats, Inbred Lew; D014162:Transfection
D015820:Cadherins; D005456:Fluorescent Dyes; C099972:H-cadherin; D034741:RNA, Small Interfering