Go live. It’s smart.SmartFlare™ RNA Detection Probes
EMD Millipore is a division of Merck KGaA, Darmstadt, Germany
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Live cell RNA detection with SmartFlare™ RNA Detection Probes
Instead of lysed cells, switch to live cells. And while you’re at it, eliminate sample preparation and transfection steps all together. Live cell RNA detection is now possible, in a single incubation step using inert nanoparticle technology to specifically detect native RNA.
And when you’re done, the probes exit the cells allowing you to perform downstream analyses with the same, unperturbed cells.
Make the smart change!SmartFlare™ Detection ProbeGAPDH expression (red) in rat astrocytes counterstained with DAPI (Blue)
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• Detecting RNA expression in live cells which gives you real-time, physiologically relevant data
• Detecting RNA in live cells in a single incubation step using inert nanoparticle technology to specifically detect native RNA.
• Saving time and resources by eliminating laborious, costly sample preparation
• Easy analysis on the fluorescent detection platform of your choice
• Non-toxic, nanoparticle-based technology that enables cells to be used for downstream assays
What’s smart
Figure 1. Uptake, bind, detect, reuse. SmartFlare™ probes enter the cell using the cell’s own endocytosis process. The probes circulate within the cell and bind to the complementary RNA sequence. This binding event releases a fluorophore, illuminating the cells for detection. Over time, the probe exits the cell, leaving the cell unchanged and free for downstream analyses.
Probe enters cell.Probe binds targetand flare releases for detection.
Probe approaches target (mRNA).
Probe exits the cell. Cell can be reused.
Capture Strand
Reporter Strand
-AAAAAAATCAACCATACACCGTGACTTTGCTTGACCC
-AGTTGGTATGTGGCACT
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Figure 2. Molecular mechanism of SmartFlare™ RNA Detection Probe
The oligo duplexes are designed with an RNA “capture” sequence and a complementary “reporter” sequence.
In absense of target, probe is quenched by gold and does
not fluoresce
When Target is present it will bind the capture strand, releasing
the reporter strand and fluorescing
Target
+
Short DuplexesFlares are dark.
Target (mRNA)
Capture Strand
Reporter Strand
Longer Duplex FormedFlare released.
-AAAAAAATCAACCATACACCGTGACTTTGCTTGACCC
-AGTTGGTATGTGGCACT
+
Short DuplexesFlares are dark.
Target (mRNA)
Capture Strand
Reporter Strand
Longer Duplex FormedFlare released.
-AAAAAAATCAACCATACACCGTGACTTTGCTTGACCC
-AGTTGGTATGTGGCACT
1. Plate cells at desired density (typical example: 30,000 cells in 200 µL complete medium in each well of a 96-well plate)
2. Dilute reconstituted SmartFlare™ reagent 1:20 in sterile phosphate-buffered saline to create working solution
3. Add 4 µL diluted SmartFlare™ reagent to each well of cells (which are 60-80% confluent)
4. Incubate overnight (16 hours) at appropriate temperature (37 °C), CO2 (5%) and relative humidity.
5. Detect fluorescence using platform of choice: flow cytometer, imaging cytometer, fluorescence microscope, etc.
Cell testing protocol (recommended protocol for testing a cell-probe combination for the first time):
• No sample prep
• No RNA amplification
• No transfection reagents
• No cell lysis
• No toxicity
RNA detection often requires laborious sample prep, RNA amplification and detection based on standard curves. SmartFlare™ RNA Detection Probes are endo-cytosed by live cells using existing cellular endocytosis machinery. Sample prep is unnecessary; simply add SmartFlare™ probes to your cultures, incubate over-night and detect the next day. Over time, the probes will exit the cell, without any adverse effects, allowing for subsequent downstream assays.
Eliminate the complexity and uncertainty associated with other RNA detection techniques. SmartFlare™ RNA Detection Probes make discovery effortless.
What’s simple
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Applications using SmartFlare™ Probes
• Track changes in RNA dynamically, over time
• Sort cell types that are traditionally difficult to isolate, using intracellular RNA markers
• Quantify miRNAs
• Assess RNA and protein in the same cellular preparations
You can take a peek at your RNA without your cells ever knowing you were there. SmartFlare™ probes enable novel experimental designs that can change how you do science. Imagine the possibilities.
Figure 3. Sorting based on intracellular RNA markers. Here cells were sorted based on miR-155 expression. miR-155 high and miR-155 low cells were isolated and then used for downstream experiments examining protein levels in the same cells.
What’s powerful
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100 101 102 103 104
Forw
ard
S ca
tter
51
102
153
204
256
105
miR-155 Expression Level (Cy5 Fluorescence)
• Reuse your SmartFlare™ probe-treated cells for downstream assays
• Live cell detection via flow cytometry
• Assess multiple RNA targets (multiplex)
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Quantify RNA using flow cytometry Quantifying RNA has never been easier. Because flow cytometry interrogates single cells for fluorescence expression, amplification is not necessary. Get specific, single cell, relative quantification of RNA in intact cells using EMD Millipore’s guava easyCyte™ or Muse™ flow cytometry platforms with SmartFlare™ Probes. Or really enhance your detection capabilities using the Amnis® Imaging Flow Cytometry systems.
Fluo
resc
ence
(M5-
510)
Cycles1 2 6 9 10 1613 19 22 25 29 4034 37314
Fluo
resc
ence
(M5-
510)
Cycles
30
40
50
60
70
80
90
100
110
40
50
60
70
80
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100
110
1 2 6 9 10 1613 19 22 25 29 4034 37314
MCF-7No probe HeLa
MCF-7 HeLa
0
20
100 101 102 103 104
40
60
80
100
120
140
EGFR mRNA probe (RED2-HLog)
FGF2 mRNA probe (RED2-HLog)
Coun
t
No probe HT1376
0
100 101 102 103 104
40
20
60
80
100
120140
160
Coun
t
HUVEC
HT1376 HUVEC
180
Figure 5.
Probe detection of mRNA levels correlates to RT-PCR data. Using SmartFlare™ technology to determine the mRNA levels of EGFR mRNA (A) in HeLa and MCF-7 cells as well as FGF2 mRNA (B) in HUVEC and HT1376 cells showed good correlation to RNA levels determined by quantitative RT-PCR.
Using live cells means you are looking at native, unamplified RNA, which can reveal meaningful links between gene expression profiles and phenotypes. Choose the way you discover what’s really happening in your cells.
Scrambled Control
MCF
-7 C
ells
HeL
a Ce
lls
EGFR Target
What’s really happening
A.
B.
Figure 4. Microscopy shows specificity and dynamic range of EGFR SmartFlare™ probe. MCF7 and HeLa cells show varying levels of EGFR expression (red) when incubated with an EGFR-specific SmartFlare™ probe. No signal is seen in cells treated with a sequence-scrambled control probe (left column).
Visualize RNA using microscopy Using SmartFlare™ RNA Detection Probes via microscopy is simple. You can see RNA target expression in living cells right under the microscope, track changes in real time, monitor gene expression profiles in mixed cell populations, and get quick yes/no answers about target expression in living cells.
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Ordering InformationProduct Catalogue No.
SmartFlare™ Develop to Order Service SF-DTO
HouseKeeping Genes
SmartFlare™ Cyclophilin A, CY5-Hu SF-139
SmartFlare™ Cyclophilin A, CY3-Hu SF-150
SmartFlare™ Cyclophilin A, CY5-Ms SF-128
SmartFlare™ Cyclophilin A, CY3-Ms SF-129
SmartFlare™ RNA Pol II, CY5 -Hu SF-130
SmartFlare™ RNA Pol II, CY3-Hu SF-131
SmartFlare™ HPRT1, CY5-Hu SF-132
SmartFlare™ HPRT1, CY3-Hu SF-133
SmartFlare™ HPRT1, CY5-Ms SF-134
SmartFlare™ HPRT1, CY3-Ms SF-135
SmartFlare™ GAPDH, CY5-Hu SF-136
SmartFlare™ GAPDH, CY3-Hu SF-126
SmartFlare™ GAPDH, CY5-Ms SF-138
SmartFlare™ GAPDH, CY3-Ms SF-125
SmartFlare™ GAPDH, CY5-Rt SF-140
SmartFlare™ GAPDH, CY3-Rt SF-141
SmartFlare™ 18S, CY5-Hu, Ms, Rt SF-142
SmartFlare™ 18S, CY3- Hu, Ms, Rt SF-143
SmartFlare™ Beta Actin, CY5 Hu SF-144
SmartFlare™ Beta Actin, CY3 Hu SF-145
Visit our website to view more performance data and browse our complete selection of ready-to-order SmartFlare™ probes or develop your own.
www.emdmillipore.com/smartflare
Product Catalogue No.
Controls
SmartFlare™ miRNA Scramble Control, CY5 SF-146
SmartFlare™ miRNA Scramble Control, CY3 SF-147
SmartFlare™ Uptake Control, CY5 SF-137
SmartFlare™ Uptake Control, CY3 SF-114
SmartFlare™ Scramble Control, CY5 SF-102
SmartFlare™ Scramble Control, CY3 SF-103
SmartFlare™ Scramble Control, CY5 + Uptake, CY3
SF-104
SmartFlare™ Scramble Control, CY3 + Uptake, CY5
SF-105
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EMD Millipore, the M mark, Muse, guava easyCyte and SmartFlare are trademarks of Merck KGaA, Darmstadt, Germany. Amnis is a registered trademark of Merck KGaA, Darmstadt, Germany.Lit No. PB4438EN00 BS-GEN-12-07299 10/2012 Printed in the USA. © 2012 EMD Millipore Corporation, Billerica MA USA. All rights reserved.
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