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Single cell RNA detection by flow cytometry gives more information than RT-PCR and can be performed on live cells Introduction Methods Understanding the role of a gene in the context of a tissue, organ or other heterogeneous cell population can be diffcult, even impossible, by using traditional RNA detection methods, which examine RNA levels in lysed, permeabilized or fxed cell samples. SmartFlare™ probe incubation: MCF-7 (Interleukin-6 (IL-6) negative) and MDA-MB-231 (IL-6 positive) human breast carcinoma cell lines incubated with Human IL-6 Cy5 SmartFlare™ probe for 16 hours at 37 °C were detached and counted. Mixtures of the two cell lines were prepared at various MCF-7:MDA-MB-231 ratios (shown in Figures 1 and 2) and analyzed along with pure cell samples. Quantitative Real-Time PCR (qRT-PCR): Total RNA was extracted from each cell sample, and subjected to quantitative real-time PCR analysis using a TaqMan® IL-6 primer set and RNA-to-Ct™ kit (Life Technologies). A Roche LightCycler® 480 real-time PCR instrument was used for amplifcation. Pure MCF-7 Cells Fluorescence History 60 Ct = ~37 55 50 45 40 35 30 25 20 12 4 6 8 10 13 16 19 22 Cycles 25 28 31 34 37 40 40 60 Ct = 30.47 ± 0.09 55 50 45 40 35 30 25 20 1 2 4 6 8 10 Our studies performed using SmartFlare™ RNA detection probes in conjunction with fow cytometry, microscopy and imaging cytometry, illustrate the value of live cell RNA detection at the single cell level to identify differences in expression levels across populations of cells. 13 16 19 22 Cycles 25 28 Confocal microscopy: Visualization of cell fuorescence was performed using a Nikon C2 confocal scanning system with 405 nm and 640 nm argon lasers. For confocal microscopy, cells were additionally stained with DAPI nuclear dye after incubation with SmartFlare™ probe. 31 34 13 16 19 22 Cycles 25 28 31 34 37 40 34 37 40 Pure MDA-MB-231 Cells Fluorescence History Fluorescence (465-510) In contrast, analyzing RNA content in individual, live cells enables identifcation of smaller cell populations that have responded to a treatment differently than the rest of the cells present. Furthermore, the ability to use those same cells for downstream experiments can enable sorting and enrichment of cells based on individual RNA levels. Fluorescence (465-510) 55% MCF-7/45% MDA-MB-231 Fluorescence History 60 Ct = 31.71 ± 0.16 55 50 45 40 35 30 25 20 1 2 4 6 8 10 Flow Cytometry: Cell samples were analyzed using a guava easyCyte™ 8HT benchtop fow cytometer to resolve cell populations based on IL-6 SmartFlare™ probe-specifc Cy5 fuorescence signal. For select samples, imaging cytometry was also performed using the Amnis ImageStream®x Mark II instrument. 75% MCF-7/25% MDA-MB-231 Fluorescence History 60 Ct = 32.23 ± 0.17 55 50 45 40 35 30 25 20 1 2 4 6 8 10 Fluorescence (465-510) Fluorescence (465-510) For example, quantitative RT-PCR, reports the average amount of RNA within a whole sample of cells and the results, while widely accepted, may not accurately refect gene expression subpopulations within heterogeneous sample settings. If a small subset of cells responds to a stimulation or treatment by increasing transcription of a certain gene, this change could easily be masked by the rest of the population when the entire sample is lysed and its total RNA is extracted. RNA detection: Pure and mixed cell samples were analyzed for to IL-6 expression using three detection methods. 37 13 16 19 22 Cycles 25 28 31 Figure 1. qRT-PCR enables detection of IL-6 mRNA in breast cancer cell lines, but cannot distinguish between high and low expressing subpopulations in heterogeneous cell samples – Cell lysates of IL-6 negative MCF-7 and IL-6 positive MDA-MB-231 cells, along with mixtures of the two cell types in the ratios shown above, were subjected to qRT-PCR analysis using an IL-6 specifc primer set. In absense of target, probe is quenched by gold and does not fuoresce Target When Target is present it will bind the capture strand, releasing the reporter strand and fuorescing + Figure 2. SmartFlare enables specifc fuorescence-based detection of RNA within live cells – SmartFlare RNA Detection Probes are gold nano-particles conjugated to duplexed oligo nucleotides. In the presence of target RNA a fuorescent reporter is released from the proximity of the gold and fuorescence can be detected. EMD Millipore is a division of Merck KGaA, Darmstadt, Germany EMD Millipore, the M mark and SmartFlare are trademarks of Merck KGaA, Darmstadt, Germany. Trademarks belonging to third parties are the properties of their respective owners. BS-GEN-13-08724 © 2013 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.