Chen Alice A, Derfus Austin M, Khetani Salman R, Bhatia Sangeeta N
Harvard-M.I.T. Division of Health Sciences and Technology/Electrical Engineering and Computer Science, Massachusetts Institute of Technology, MA, USA.
Nucleic Acids Res. 2005 Dec 13;33(22):e190. doi: 10.1093/nar/gni188.
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.
利用RNA干扰来确定基因型/表型相关性的一个关键问题是细胞群体内基因沉默的一致性。转染效率、递送诱导的细胞毒性以及高浓度小干扰RNA（siRNA）的“脱靶”效应的差异会混淆功能研究的解释。为了解决这个问题，我们开发了一种监测siRNA递送的新方法，该方法将未修饰的siRNA与半导体量子点（QD）结合作为多色生物探针。我们使用标准转染技术将siRNA与QD共转染，从而利用光稳定的荧光纳米颗粒来追踪核酸的递送，根据转染程度对细胞进行分类并纯化均一沉默的亚群。与其他RNA干扰追踪方法（共递送报告质粒和对siRNA进行末端标记）相比，量子点在广泛选择的非重叠颜色方面表现出卓越的光稳定性和可调光学特性。因此，这个简单的模块化系统可以扩展到多重基因敲低研究，如在针对两个生物学靶点的双色原理验证研究中所示。当该方法应用于研究T-钙黏蛋白（T-cad）在细胞间通讯中的功能作用时，需要通过量子点标记获得的高度沉默细胞亚群来观察基因敲低的显著下游效应。
