SmartFlareTM for live cell RNA detection by using flow cytometry Erwin Swart Cellular Specialist BeNeLux Merck Millipore Current RNA Detection Methods Until now detecting RNA in cells required: Lyse cells,RNA Extraction, cDNA Synthesis, Amplification Transfection Fixation\Permeabilization We asked what if we could: a) Analyse RNA directly in living cells b) Re-use cells in down stream experiments We came up with a smarter idea... 2 A Smarter Idea Novel technology that could revolutionize genomic and cell biology applications Smart Powerful •Live cell detection •Detect Native RNA •Non toxic reagent •Re-use your cells •Sorting •Multiple biomolecules Simple •Mix and read •No sample prep •Open detection platform 3 We call it SmartFlare Live Cell RNA Detection using Nanoparticle Technology 4 How does it work? Single incubation, single reagent, overnight detection Using the cells own machinery 5 1. SmartFlare enters the cell 2. Detects the RNA of interest 3. Exits the cell allowing further experimentation Workflow SmartFlare protocol is a simple mix and read 1. Reconstitute & Dilute 7 2. Add to cells in culture Allow to incubate overnight 3. Detect RNA levels via microscopy or Flow Some Detail on Controls for Experiments Uptake Control: - SmartFlare Entry into the cell Housekeeping control: - Cell health - Did the experiment work? - Inter-experiment comparisons Scramble Control: Background due to - SmartFlare degradation - Unspecific probe release miR-155 detected by SmartFlare Confocal microscopy Uptake Scramble miR-155-5p A B C D E F HeLa HUVEC Detection using Microscopy Visualize both miRNA and mRNA in live cells Bright Field Housekeeping 18S Cy3 in B35 Neuroblast Cells SmartFlare miR-21 Cy5 In DU145 Cells EGFR Cy5 in HeLa cells Overlay 6 SmartFlare Target Specificity in Co-Culture Co-cultured rat neurons and astrocytes show specificity for their respective markers Beta III Tubulin (Neuron) SmartFlare Scrambled SmartFlare Cy5 (Target) Brightfield Vimentin (Astrocyte) SmartFlare 10 Astrocytes Neurons Neurons Astrocytes Protein and mRNA in the same cells Dual Detection of mRNA and Protein by Microscopy. DAPI GFAP Protein GFAP mRNA Rat Primary Astrocytes SmartFlare Effects on Cellular State C. lane 1 EGFR SF B. Scramble SF A. Unflared SmartFlare is non-toxic, does not alter gene expression or translation. 2 3 250 (kDa) 150 (kDa) blot: EGFR(~180kDa) 1 2 3 250 (kDa) A. Cellular viability not affected by SmartFlare addition B. Genome wide arrays showing no significant change to gene expression following the addition of SmartFlare. C. Translation does not seem to change following SmartFlare detection by Western Blot CBB 150 (kDa) Normalized signal intensity =Blot/CBB EGFR SmartFlare in HeLa cells SmartFlare Sensitivity and Correlation Data Twist -1Targets quantified by Smartflare & Flow correlate to RT-PCR qRT-PCR ct 30.61 MCF7 ct 24.72 HS578T Low Expression in MCF-7 Cells High Expression in Hs578t cells Flow Data MFI 137.36 MCF7 MFI 624.01 HS578T 9 Single Cell Resolution of SmartFlare by Flow Allows for identification of divergent cell populations not detectable by qRT-PCR Il-6 expression by SmartFlare Il-6 expression by qRT-PCR MDA 100% MDA-MB231 MCF-7 100% MCF-7 MDA MCF-7 Ct = 30.47 ± 0.09 Ct = ~37 Ct = 31.71 ± 0.16 45% MDA-MB-231/ 55% MCF-7 siRNA Gene Modulation Detection SmartFlare accurately quantifies knockdown of genes using siRNA SmartFlare Survivin Probe Detection by Flow Following siRNA LNCaP cells w/o Flare LNCaP cells + Survivin siRNA + Survivin Flare LNCaP cells + Survivin Flare SmartFlare Detection of Gene Expression Modulation Correlates with qRT-PCR 69% gene knockdown 65% gene knockdown Sorting using SmartFlare Probes Sort cells based on RNA then perform downstream assays HeLa HUVEC Mixed cell populationsorted by miR-155 expression 14 Antibody staining for VCAM in sorted populations Cells which expressed miR-155 from sort are expressing VCAM as expected. SmartFlare Visualized via Amnis Imaging Flow EGFR RNA and Protein Expression in SKBR cells Overlay Bright Field EGFR SmartFlare EGFR Antibody Hoechst SKBR-3 Cells Overlay Bright Field Scramble SmartFlare EGFR Antibody Hoechst SKBR-3 Cells Flow cytometric imaging – Amnis® ImageStream Dual Detection of EGFR protein (immunostaining) and mRNA (SmartFlare) MCF-7 Anti-EGFR EGFR SmartFlare SKBR3 Bright Field Anti-EGFR Merge EGFR SmartFlare Bright Field Merge Co-detection of EGFR mRNA and cell surface EGFR in low- and high-expressing breast cancer cells with Guava easyCyte 8HT Red2: Scrambled Cy5 SmartFlare Green: Alexa488-msIgG1 Isotype 1.78 0.00 Red2: hEGFR Cy5 SmartFlare Green: Alexa488-anti-total EGFR 0.90 98.44 SKBR3 Alexa488 antibody fluorescence 97.56 0.60 0.16 4.91 1.59 0.49 11.74 0.00 MCF-7 98.06 0.35 SmartFlare fluorescence 44.26 39.09 Equipment for Detection of SmartFlare™ probes Fluorophore IndoCarbocyanine Cy3 IndoCarbocyanine Cy5 Excitation Peak 550 650 Emission Peak 570 670 8HT / 6HT-2L Summery – Cellular Detection SmartFlare is a novel Live Cell RNA Detection Technology Non-toxic Does not alter gene expression Does not require transfection reagent Mix and read easu of use The probes give you more informationthan typical detection methods Live cell dynamic tracking of RNA levels Ability to understand the RNA expression level at single cell level Monitor multiple biomolecules simultanously Many unique applications Study RNA levels during stimulations understanding changes in real time Sort cells based on any RNA expression level Re-use cells which have been profiled for their RNA expression levels Determine kn ockdown efficiency and enrich for 100% knockdown Sort cells following stimulations to enrich for responders Two Smart Solutions For RNA Detection SmartRNAplex™ MicroRNA Profiling Assay SmartFlare™ RNA Detection Probes Find the miRs that matter Live cell RNA Detection SmartFlare Probes vs SmartRNAplex Assay SmartFlare SmartRNAplex mRNA and miRNA miRNA only Live Cell Culture Crude Digests & Purified RNA Plex 1-2 targets (Knows their miR) 10 – 68 targets (Doesn’t know their miR) Detection Platform Flow Cytometry & microscopy Flow Cytometry RNA Type Sample Type Questions? Merck Millipore Booth 7C021 – Hal 7 A Smarter Idea for Multiplexing miRNAs Smart Simple Powerful • Multiplex up to 68 different miRNAs across up to 96 samples • Analyze Crude Digests & Purified RNA • Less than 30 mins of hands on time • Minimal sample prep • Easy detection using flow cytometry • More plex than RT-PCR • More samples than microarray Introducing SmartRNAplex™ MidPlex MiRNA profiling assay via flow cytometery • • • Total assay time ~ 3 hours < 30 minutes hands-on time Process up to 96 samples at once SmartRNAplex™ Technology Overview How it works 3 Steps: 1. Hybridize: target binds to complementary probes on hydrogel particles 2. Label: Universal, biotinylated label is ligated only to correctly bound targets 3. Report: Fluorescent reporter binds to biotinylated label for detection via flow cytometry 2 7 SmartRNAplex™ Assay Overview Standard Flow Cytometer Firefly™ Software Bound Particles OR Muse Cell Analyzer Questions and contact Information ? Jan Brants, PhD Technical Sales Specialist Merck Millipore E-Mail: [email protected] Tel: 0900 040 16 26 Appendix Some Detail on Controls for Experiments 15 SmartFlare Early in-vivo Work 12 SmartFlare does not alter gene expression Genome wide arrays showing no significant change to gene expression following the addition of SmartFlare. Genome wide arrays (> 47,000 transcripts) MDA-MB-231 adenocarcinoma cells, mammary gland and epithelial cells : -w/o SmartFlare, Scrambled SmartFlare, Targeted SmartFlare (EGFR), triplicates; analyzed by core informatics group. No detectable difference three groups. ( Core facility called back: mistake - no difference in samples).  SmartFlare not affect gene expression. (After 16h, Illumina platform) Extracellular detection in the lower fmolar range Standard curve generated with synthetic oligos show promise for serum detection of secreted miRNA. Co-detection of EGFR mRNA and cell surface EGFR in low- and high-expressing breast cancer cells with Guava easyCyte 8HT Red2: Scrambled Cy5 SmartFlare Green: Alexa488-msIgG1 Isotype 1.78 0.00 Red2: hEGFR Cy5 SmartFlare Green: Alexa488-anti-total EGFR 0.90 98.44 SKBR3 Alexa488 antibody fluorescence 97.56 0.60 0.16 4.91 1.59 0.49 11.74 0.00 MCF-7 98.06 0.35 SmartFlare fluorescence 44.26 39.09 SmartFlare V RT PCR Large fold changes captured by RT are much smaller by SmartFlare Target Eralpha APRIL RT ct value 37 30 Scramble MFI 15 29 neg cell line Uptake MFI 21 17 SmartFlare MFI 95.77 128 RT ct high value 22 26 Scramble MFI 33 98 pos cell line Uptake MFI 20 26 SmartFlare MFI 226 364 MFI fold change 2.51 1.76 RT fold change 5000 9.48 ct difference 15 4 38 Presentation title in footer | 00 Month 0000 Arial 8pt SmartFlare EGFR expression levels trend similarly to RT-PCR values A. B. Figure 1. Correlation between EGFR mRNA by qRT-PCR and SmartFlare live-cell RNA detection probe in breast cancer cells. 4 breast cancer cell lines, T47D, MCF7, SKBR3 and MDA-MB-231, known to vary in EGFR expression, were analyzed for EGFR mRNA . A, live cells were incubated overnight with Cy5-labeled SmartFlare probes for scrambled control or EGFR, and analyzed by flow cytometry on the guava easyCyte 8HT. Ratio of mean fluorescence intensity of EGFR to scrambled is shown. B, cells were lysed and subjected to qRT-PCR for EGFR and GAPDH mRNAs. Live-cell analysis yielded relative expression ranking of MDA-MB-231>SKBR-3>T47D>MCF7, which correlated well with the relative expression observed with qRT-PCR. Single Cell Resolution of SmartFlare by Flow Allows for identification of divergent cell populations not detectable by qRT-PCR Il-6 expression by SmartFlare Il-6 expression by qRT-PCR MDA MFI = 744.81 MCF-7 100% MDA-MB231 100% MCF-7 Ct = 30.47 ± 0.09 Ct = ~37 MFI = 168.77 MDA MCF-7 MFI = 323.48 Ct = 31.71 ± 0.16 45% MDA-MB-231/ 55% MCF-7 Correlating IL-6 mRNA levels observed by qRT-PCR with SmartFlare MFI IL-6 Hu-Cy5 SmartFlare MFI (RED2) 800 41 700 600 500 y = 5,9079x + 76,381 R² = 0,8865 400 300 200 100 0 0 20 40 60 80 100 Normalized percentage of IL-6 mRNA by qRT-PCR Presentation title in footer | 00 Month 0000 Arial 8pt 120 % of IL6 expressing cells (MDAs) correlated tighter than the RT PCR IL-6 Hu-Cy5 SmartFlare MFI (RED2) IL-6 SmartFlare Cy5 MFI in samples with varying percentages of MDA-MB-231 cells 42 800 700 600 500 400 y = 5,8325x + 115,42 R² = 0,9488 300 200 100 0 0 20 40 60 80 % of MDA-MB-231 (IL-6 high) cells in sample Presentation title in footer | 00 Month 0000 Arial 8pt 100 Extracellular detection of miRNA shows linearity at low fMol range