e190 Nucleic Acids Research, 2005, Vol. 33, No. 22 PAGE 6 OF 8 Figure 5. Fluorescence/phase micrographs of two color QD transfections. (A) Low-gated cells (L++, where ++ indicates the presence of two colors of QD) nearly lack orange or green QDs. (B) High-gated cells (H++) fluoresce brightly with punctate green and orange QDs (enlarged in inset). Scale bars 100 mm. (30% protein knockdown) from a highly transfected popula- tion (95% knockdown). In the highest 8% of the dual color, dual siRNA co-transfected cell population, highly effective silencing of both Lmna gene (96% knockdown) and T-cad gene (98% knockdown) was achieved. Given the wide spec- trum of QD color possibilities, this method promises to be useful for tracking and sorting multiple siRNA-mediated knockdowns within one cell population. The utility of RNAi as a functional genomics tool is pre- dicated upon associating gene silencing with downstream phenotypic observations. Yet non-uniform gene silencing may obscure bulk measurements (protein and mRNA) com- monly used to validate gene knockdown and obscure geno- type/phenotype correlations. We compared the downstream effects of non-uniform and homogenous gene silencing to speciﬁcally examine the stabilizing effect of non- parenchymal cells (3T3 ﬁbroblasts) on hepatocellular function in vitro (36). Recently, several cadherins from hepatocyte- ﬁbroblast junctions were identiﬁed as potential mediators of liver-speciﬁc function in vitro (37). Based on this ﬁnding, we transfected ﬁbroblasts with T-cad siRNA or T-cad siRNA/QD complexes, sorted each population according to high or low cellular ﬂuorescence, and co-cultivated the populations with hepatocytes. Markers of liver-speciﬁc function, albumin syn- thesis and cytochrome P450 1A1 (CYP1A1) activity, were measured in hepatocyte/3T3 co-cultures (Figure 6). Compared to control co-cultures, signiﬁcant down-regulation in hepato- cellular function (2-fold) was observed exclusively in the cul- tures that had been treated with T-cad siRNA/QD complexes and sorted for high cellular ﬂuorescence. These studies implic- ate a role for ﬁbroblast T-cad protein expression in modulating hepatocellular function in vitro, an interpretation revealed only once a homogenously-silenced population of ﬁbroblasts was obtained. In this work, we have identiﬁed a role for T-cad in cell–cell communication between hepatocytes and non-parenchymal cells using a technically simple method for enriching trans- fected cells which can be applied to further studies. Attempts to improve silencing by simply using higher concentrations of siRNA do not improve knockdown but may actually negat- ively regulate RNAi-mediated gene silencing (Supplementary Figure S1) (40,41). In addition, excesses of either siRNA or cationic liposome has been shown to induce increased cyto- toxicity, interferon response (42) and ‘off-target’ effects (43). While current techniques are available to ﬂuorescently track Figure 6. Significant downstream gene knockdown effects of T-cad gene silencing are observed only in a homogenous silenced cell population. Murine 3T3 fibroblasts transfected with T-cad siRNA alone or with T-cad siRNA/QD complexes were FACS-sorted for low 10% (L) or high 10% (H) intracellular fluorescence. Symbols and + indicate the absence or presence of QD during transfection. To study the stabilizing effect of non-parenchymal cell (3T3 fibroblast) protein expression on liver-specific function, control or trans- fected/sorted 3T3 cells were added to hepatocyte cultures 24 h after hepatocyte seeding. Liver-specific function was assayed by measuring albumin synthesis and cytochrome P450 1A1 (CYP1A1) activity of cultured media sampled at 72 and 96 h after 3T3 seeding and averaged. Error bars represent standard error of the mean (n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001 (One-way ANOVA statistical analysis test). RNAi delivery (co-delivery of ﬂuorescent protein expressing plasmids, end-labeling with a dye), none offer such a simple, modular and versatile means to generate a homogeneously silenced population. In addition to superiority over dyes in brightness and photostability (Supplementary Figure S2), multi color QD labels can be used interchangeably, with all particle sizes employing a common, passive mechanism to form delivery complexes. The point at which a cell’s RNAi machinery saturates is not yet known; yet at the least this capability simpliﬁes known strategies to enrich for dual gene knockdowns using antibiotic-resistant markers that are co-transfected, transcribed and selected. The numerous non- overlapping color possibilities of QDs (12 commercially avail- able in the spectral range of FACS detectors) may further enable multiplexed and combinatorial gene studies for the