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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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.

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510)

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MCF-7No probe HeLa

MCF-7 HeLa

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100 101 102 103 104

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EGFR mRNA probe (RED2-HLog)

FGF2 mRNA probe (RED2-HLog)

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HT1376 HUVEC

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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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For other countries across Europe and the world, please visit: www.emdmillipore.com/offices

For Technical Service, please visit: www.emdmillipore.com/techservice

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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.