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Furthermore, we showed that SmartFlare™ RNA detection technology could be used to study gene expression without compromising the integrity or the viability of individual cells. This capability enabled downstream experimentation and correlation of gene expression to cell behavior. References 1. Context-specifc regulation of NF-kB target gene expression by EZH2 in breast cancers. , Lee ST, Li Z, Wu Z, Aau M, Guan P, Karuturi RKM, Liou YC & Yu Q, Mol Cell 43 (2011) 798-810. 2. Interleukin-6 is a potent growth factor for ER-α-positive human breast cancer, Sasser AK, Sullivan MJ, Studebaker AW, Hendey LF, Axel AE & Hall BM, FASEB J Res Comm 21 (2007) 3763-3770. Visit our website to view more performance data, browse our complete selection of ready-to-order SmartFlare™ probes, or design your own. www.millipore.com/smartfare Forward Scatter 1000 3000 5000 7000 9000 0 20 40 60 80 104 103 101 102 IL-6 Hu Cy5 SmartFlare™ 104 0 20 40 60 80 101 102 103 104 IL-6 Hu Cy5 SmartFlare™ 0 20 40 60 80 Forward Scatter 1000 3000 5000 7000 9000 Count 120 160 200 100 101 102 103 104 IL-6 Hu Cy5 SmartFlare™ 0 20 40 60 80 Forward Scatter 1000 3000 5000 7000 9000 Count 120 160 200 100 800 700 600 500 400 300 200 100 0 100 101 102 103 104 IL-6 Hu Cy5 SmartFlare™ IL-6 SmartFlare™ Cy5 MFI in Samples with Varying Percentages of MDA-MB-231 B. IL-6 Hu-Cy5 SmartFlare™ MFI (RED2) 101 102 103 104 IL-6 Hu Cy5 SmartFlare™ Forward Scatter 1000 3000 5000 7000 9000 Count 120 160 200 103 101 102 IL-6 Hu Cy5 SmartFlare™ 100 Summary Many focus areas of cell biology, such as immunology and cancer research, require the resolution of cell subpopulations within heterogeneous samples based on gene expression levels. For example, efforts to elucidate genetic bases of tumor progression have been greatly challenged by tumor heterogeneity and cellextrinsic effects. Thus, ability to specifcally detect RNA in single, live cells may generate more biologically signifcant gene expression data by identifying variations in expression levels across subpopulations within a cell sample. 104 100 y = 5.8325x + 115.42 R2 = 0.9488 20 40 60 % of MDA-MB-231 (IL-6 high) cells in sample 1 80 100 A. DIC SmartFlare™ Cy5 Merged DIC SmartFlare™ Cy5 Merged MDA-MB-231 (IL-6 High) We analyzed SmartFlare™ probe-mediated fuorescence in our breast cancer cell samples using fow cytometry in order to assess IL-6 mRNA expression. Just as with qRT-PCR, we clearly observed the differences in expression levels between MCF-7 and MDA-MB-231 cell types. However, while qRT-PCR only provided information about averaged IL-6 expression levels, SmartFlare™ technology enabled us to study the variation in expression within the mixed cell samples on a single cell level. Specifcally, we observed and quantifed the distribution of single cell events in the Cy5 low and Cy5 high gates in the heterogeneous cell mixtures, representative of MCF-7 and MDA- 101 102 103 IL-6 Hu Cy5 SmartFlare™ 100 100 In this study, we have demonstrated the use of SmartFlare™ technology to identify cell subpopulations in live heterogeneous mixtures based on IL-6 mRNA expression, a determination that cannot be achieved through conventional RNA detection techniques. 104 MCF-7 (IL-6 Negative) In order to more effectively resolve single cells in our breast cancer cell mixtures based on differences in their gene expression, we analyzed our cell samples using an IL-6 Cy5 SmartFlare™ RNA detection probe. SmartFlare™ technology enables relative quantifcation of specifc RNA sequences using fuorescent detection platforms. Furthermore, treatment of cells with SmartFlare™ probes does not compromise their integrity or viability, allowing for live cell analysis and downstream experimentation. 103 101 102 IL-6 Hu Cy5 SmartFlare™ 100% MDA-MB-231 SmartFlare™ technology enables single-cell analysis of IL-6 mRNA expression in breast cancer cell mixtures by fow cytometry SmartFlare™ technology enables specifc detection of RNA species in single cells without compromising their integrity or viability. We demonstrated this by subjecting MCF-7 and MDA-MB-231 cells to confocal microscopy and imaging cytometry after incubation with IL-6 Cy5 SmartFlare™ probe. In the confocal micrographs (Figure 4), both cell types retained their normal morphologies, even in the presence of probes, and the differences in Cy5 fuorescence between the two cell types could be easily distinguished. Meanwhile, imaging cytometry allowed for more in-depth correlation between qualitative and quantitative resolution of MCF-7 and MDAMB-231 cells based on IL-6 expression. 100 55% MCF-7 45% MDA-MB-231 In order to more effectively resolve single cells in our breast cancer cell mixtures based on differences in their gene expression, we analyzed our cell samples using an IL-6 Cy5 SmartFlare™ RNA detection probe. SmartFlare™ technology enables the specifc quantifcation of RNA sequences using fuorescent detection platforms. Furthermore, treatment of cells with SmartFlare™ probes does not compromise their integrity or viability, allowing for live cell analysis and downstream experimentation. Differences in IL-6 mRNA levels in breast cancer cell lines can be further analyzed visually in SmartFlare™ treated cells through confocal microscopy and imaging cytometry Figure 3. IL-6 Cy5 SmartFlare probe detects IL-6 mRNA levels in MCF-7 and MDA-MB-231 cell samples, while also identifying and resolving subpopulations based on gene expression – Pure and mixed cell samples are subjected to fow cytometry analysis following 16 hr incubation with IL-6 Cy5 SmartFlare. (A) MCF-7 and MDA-MBA-231 cells can be clearly identifed and quantifed in cell samples based on RED2 channel fuorescence intensity, particularly when using a dot plot to visualize cell populations. (B) As the percentage of MDA-MB-231 cells increases in the mixed sample, higher overall Cy5 mean fuorescence is detected. 75% MCF-7 25% MDA-MB-231 We aimed to simulate experimental settings where the expression of an mRNA is heterogeneous within a cell sample. We used MCF-7 and MDA-MB-231 breast cancer cell lines, which vary signifcantly in their expression of IL-6 mRNA1,2, and subjected pure and mixed samples of these cells to different RNA detection technologies. First, we examined lysates generated from cell samples for IL-6 expression using qRT-PCR. As expected, we observed a signifcant difference in expression between the pure MCF-7 cells (IL-6 negative) and MDA-MB-231 cells (IL-6 positive), with a gradient of expression levels in the mixed cell samples depending on the percentage of MDA-MB-231 cells present. However, we were not able to resolve single cells or subpopulations based on mRNA levels. Note that in Figure 1, a near 50/50 mixture of IL-6 positive and negative cell types yields a Ct value that is similar from the Ct value from a pure population of IL-6 negative cells. MB-231 cells, respectively, while also gaining information about overall IL-6 mRNA expression. 100% MCF-7 Quantitative real-time PCR is effective in determining overall levels of IL-6 mRNA in mixed cell samples, but is limited in its ability to resolve subpopulations of varied expression. Count 120 160 200 A. Results B. Focused Brightfeld Cy5 IL-6 Low Population (MCF-7) IL-6 High Population (MDA-MB-231) Figure 4. Visual detection of IL-6 mRNA fuorescence (A) MCF-7 and MDA-MB-231 cells were incubated with IL-6 Cy5 SmartFlare for 16 hours, stained with Hoechst 33342 DNA dye, and subjected to confocal microscopy. The two cell types can be distinguished both by morphological differences as well as distinctive Cy5 fuorescence relative to their IL-6 mRNA expression. (B) When analyzing a mixed MCF-7 / MDA-MB-231 cell sample using an Amnis ImageStream®x Mark II imaging cytometer, one can correlate subpopulations of high and low Cy5 fuorescence with images of individual cells contained within these populations.