Thermo Scientific Dharmacon miRIDIANmicroRNA Mimics & Hairpin Inhibitors

Highly potent oligonucleotides for modulating miRNA function

Reliable reagents for reproducible studies of miRNAs

Advanced design for all human, mouse and rat miRNAs

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Supplement miRNA function in cells with LOW endogenous levels

Inhibit miRNA function in cells with HIGH endogenous levels

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Add miRNAMimics

Add miRNAHairpin Inhibitors

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Thermo Scientifi c Dharmacon miRIDIAN microRNA Mimics and Hairpin Inhibitors: modulation of an endogenous RNAi mechanism

Transcribed pri-miRNA is cleaved by Drosha-DGCR8 protein complex to form a hairpin pre-miRNA.Pre-miRNA is transported out of the nucleus by an Exportin 5 protein complex.Pre-miRNA is cleaved by Dicer to form a short double-stranded miRNA duplex.miRNA duplex is loaded into RISC. Cleavage of inactive strand results in formation of programmed RISC.Programmed RISC binds to 3’-UTR of target mRNA (5/5a). miRNA inhibitors compete with mRNA for programmed RISC (5b).Target protein expression is decreased when miRNA mimic is added (6a) and increased when miRNA inhibitor is added (6b).

Effects of miRNA mimics and inhibitors on protein expression

microRNAs are endogenous non-coding ~22mer RNAs that are highly conserved and regulate gene transcript levels through RNA interference mechanisms. As key players in the fi ne-tuning of biological networks, microRNAs have signifi cant diagnostic and prognostic potential as biomarkers not only of fundamental cellular physiology but also of disease etiology and progression. To explore microRNA biology, we provide potent and specifi c tools for dissecting microRNA function.

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microRNA experimental workfl ow

Supplementing or inhibiting microRNA activity and examining the resulting phenotypic effects is crucial for understanding basic microRNA involvement in, for example:

RNA interference mechanisms

Fundamental cellular and physiological processes

Gene regulatory networks and pathways

Disease etiology and progression

Response to disease treatment regimens

Effects of miRNA mimics and inhibitors on protein expression

• microRNA Expression Profi ling Services - Global microRNA expression analysis through the Thermo Scientifi c RNAi Discovery and Therapeutic Services (RDTS)

• Mimic and Hairpin Inhibitor library screening services - End-point and high-content assay read-outs

• miRIDIAN microRNA Mimics and Hairpin Inhibitors - Individual molecules and plated libraries

• miRIDIAN microRNA Mimic and Hairpin Inhibitor Experimental Controls

• Thermo Scientifi c DharmaFECT Transfection Reagents

Product and services for the microRNA experimental workfl ow

DISCOVERY

DETECTION

MODULATION

TARGET ID

miRIDIAN microRNA Mimics effectively simulate miRNA function

Optimization of miRIDIAN microRNA Mimic design

miRIDIAN microRNA Mimic function for 11 human miRNAs was assayed in HeLa and HepG2 cells 48 hours after transfection of 10 nM mimic using a dual luciferase reporter system and normalized to the control (no mimic). Similar results were obtained with 1 nM miRIDIAN microRNA Mimic (data not shown).

miRNA mimic function was assayed in HeLa cells 24 and 48 hours after transfection of 10 nM miR-375 native-based mimic, modifi ed native-based mimic, or miRIDIAN microRNA Mimic using a dual luciferase reporter system and normalized to the control (no mimic).

1 http://microrna.sanger.ac.uk

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Dharmacon® miRIDIAN microRNA Mimics

miRIDIAN microRNA Mimics are double-stranded RNA oligonucleotides, chemically enhanced with a proprietary design and available for all human, mouse and rat miRNAs in the miRBase Sequence Database.1

Supplement miRNA activity with miRIDIAN microRNA Mimics to study gain-of-function effects

Superior performance in comparison to native-based mimic design

Effective mimic of endogenous mature miRNA function

Preferential programming of RISC-like complex with active strand of miRNA andexclusion of passenger strand through a proprietary chemical modifi cation pattern

specifi c programming of RISC

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Time course of inhibition by three different designs targeting endogenous miR-23 in a MCF-7 reporter cell line from a stably-integrated luciferase construct. miRIDIAN Hairpin Inhibitor, mature antisense (AS) and Competitor E inhibitors were transfected at 40 nM using DharmaFECT ®1. Data was normalized to cells with no inhibitor, so no inhibition is equal to 1.

* Patent pending1 http://microrna.sanger.ac.uk 2 This novel microRNA inhibitor design is based on fi ndings published by Vermeulen et al. (Double-stranded regions are essential design components of potent inhibitors of RISC function. RNA, May 2007. 13: 723 - 730).

Six co-expressed miRNAs (mir-17-5p, miR-18a-5p, miR-19a, miR-20, miR-19b-1 and miR-92-1) from the "Cancer Cluster" were inhibited by miRIDIAN Hairpin Inhibitors. All six miRIDIAN Hairpin Inhibitors were pooled together for a 0.8 nM total inhibitor concentration. Pools were co-transfected with DharmaFECT Duo into HeLa cells with one of six reporter plasmids specifi c for each miRNA. Data were normalized to similarly-treated empty reporter plasmid only.

miRIDIAN microRNA Hairpin Inhibitors* are potent and long-lived at low concentrations

miRIDIAN microRNA Hairpin Inhibitors allow combinatorial inhibition of clustered miRNAs

Dharmacon miRIDIAN microRNA Hairpin Inhibitors

miRIDIAN microRNA Hairpin Inhibitors2 are single-stranded, chemically enhanced oligonucleotides available for all human, mouse and rat miRNAs in the miRBase Sequence Database.1

Most effective inhibition of endogenous mature microRNA function by means of innovative design

Patent-pending molecule combines chemical modifi cations and completely novel secondary structure motif

Superior potency and longevity in comparison to any other synthetic product offered commercially

Enhanced potency and longevity allows for multiplexed microRNA inhibition at very low nanomolar concentrations with minimal toxicity

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miRIDIAN microRNA Mimic and Hairpin Inhibitor Negative Controls

miRIDIAN microRNA Negative Control sequences are based on C. elegans miRNAs (#1: cel-miR-67, #2: cel-miR-239b) for use as negative experimental controls in human, mouse and rat cells. The Negative Controls have been analyzed by BLAST against all human, mouse and rat genomic sequences and miRNA sequences in the miRBase Sequence Database.1

Non-targeting mimics and inhibitors for use as controls for non-sequence-specifi ceffects in miRNA experiments

Allow distinction between mimic or inhibitor activity and background effects

Designs and modifi cations are identical to experimental miRIDIAN microRNA Mimics and Hairpin Inhibitors

No identifi able effects on tested miRNA function in multiple cell lines

miRIDIAN Hairpin Inhibitor Negative Controls are tools for distinguishing between miRNA-specifi c and non-specifi c effects

Effects of miRIDIAN microRNA Hairpin Inhibitor Negative Controls on the function of miR-21 were assayed at 48 hours after transfection of 5 nM inhibitor or negative control in H9c2, NIH3T3 or HeLa cells using DharmaFECT Duo and a dual luciferase reporter system.

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Effects of miRIDIAN microRNA Mimic Negative Controls on the function of three human miRNAs were assayed at 24 hours after transfection of 10 nM mimic or negative control in HeLa cells using a dual luciferase reporter system.

1 http://microrna.sanger.ac.uk

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miRIDIAN microRNA Mimic Positive Controls

Successful microRNA functional studies begin with optimization of the assay in a cell line and or type of interest. The miRIDIAN microRNA Mimic Housekeeping (HKG) Positive Controls provide the ability to monitor function of a mimic molecule at the mRNA level which can be assessed using standard transcript quantifi cation methods such as qRT-PCR.

The miRIDIAN microRNA Mimic Endogenous Positive Control provides the ability to monitor effects of a specifi c mimic on target protein levels in a validated endogenous assay. The assay is based upon the targeted activity of miR-122 on Aldolase A mRNA levels in cell lines that express low to moderate levels of miR-122.

Validated miRIDIAN microRNA Mimic that targets Aldolase A in human, mouse and rat

Provides the ability to optimize assay conditions by monitoring mimic functionon an endogenous gene target (Aldolase A) with a conserved miR-122 binding site

Employ the same structure and design as experimental miRIDIAN microRNA Mimics

Target the 3’ UTR (untranslated region) of standard housekeeping genes, PPIB or GAPDH

Allow a clean, straightforward cleavage-based readout of mimic function

Many cells lines express low to moderate levels of miR-122. Aldolase A is a predicted target of miR-122 and the 3‘ UTR is conserved in human, mouse and rat at the 8-mer miR-122 predicted seed site. miRIDIAN microRNA Mimic designed to modulate endogenous miR-122 was transfected at 50 nM (Huh-7 at 40 nM) using DharmaFECT 1 into the indicated cell lines and assessed for their ability to decrease AldoA mRNA levels. AldoA down-regulation was determined using the Quantigene branched DNA assay (Panomics) at 3 days (HepG2 at 5 days) post-transfection.

miRIDIAN microRNA Mimic Endogenous Control leads to regulation of Aldolase A in human, mouse and rat cell lines

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miRIDIAN microRNA Hairpin Inhibitor Positive Controls

The miRIDIAN microRNA Hairpin Inhibitor Positive Control targets a well-conserved microRNA (miR-16) that is broadly expressed in many human, mouse and rat cell lines. This positive control allows the optimization of microRNA functional assays and evaluation of hairpin inhibitor function.

2 nM of miR-16 hairpin inhibitor is suffi cient to bind or sequester a majority of endogenous miR-16

Endogenous levels of miR-16 were assayed 48 hours after transfection of either miRIDIAN miR-16 Hairpin Inhibitor, miR-21 Hairpin Inhibitor or Hairpin Inhibitor Control #1. HeLa cells were transfected using DharmaFECT 1.

Detection of miR-16 by qRT-PCR was performed using a poly-A tailing method (Shi R and Chaing VL “Facile means for quantifying microRNA expression by real-time PCR” (Biotechniques 39:519-529 October 2005 ) and Thermo Scientifi c Verso SYBR Green 2-Step QRT-PCR Kit (ABgene) according to protocol. Relative expression of miR-16 fi rst normalized by miR-21 expression and then further normalized to miRIDIAN Hairpin Inhibitor Control #1 levels.

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Targets miR-16 in human, mouse and rat cells resulting in reduced miR-16 activity

Allows the optimization of microRNA inhibition assays by targeting an endogenous microRNA in a cell line which expresses miR-16

Demonstrates specifi c inhibition of miR-16 which can be directly assessed by qRT-PCR and/or Northern blotting

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Dye-labeled miRIDIAN Mimic and Hairpin Inhibitor Transfection Controls allow for qualitative evaluation of transfection

validated experimental controls

miRIDIAN microRNA Mimic and Hairpin Inhibitor Transfection Controls

The miRIDIAN microRNA Mimic and Hairpin Inhibitor Transfection Controls are Dy547-labeled Mimic and Hairpin Inhibitor that are based on C. elegans miRNA (#1: cel-miR-67) for monitoring delivery into human, mouse and rat cells.

Non-targeting miRIDIAN Mimic and Hairpin Inhibitor labeled with Thermo Scientifi c Pierce Dy547 (absorbance/emission max: 557/570 nm)

Transfection can be monitored and visualized by epifl uorescence

Control designs and modifi cations are identical to experimental miRIDIAN microRNA Mimics and Hairpin Inhibitors

No identifi able effects on tested miRNA function in multiple cell lines

Dye-labeled miRIDIAN Mimic and Hairpin Inhibitor Transfection Controls allow for qualitative evaluation of transfection

Images shown are of human (left), rat (middle) and mouse (right) cells 48 hours after transfection with Pierce Dy547-labeled miRIDIAN Mimic Negative Control #1 (top panels) and miRIDIAN Hairpin Inhibitor Negative Control #1 (bottom panels). Cells were counterstained with Hoechst 33342 (blue, nuclei).

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HairpinInhibitor

H9c2 C2C12

Specifi c microRNA activity through use of chemical modifi cations to induce strand-bias

Potent and long-lasting microRNA inhibition demonstrated up to 14 days or longer

Validated negative controls to distinguish non-specifi c effects

Validated positive controls for assay development and optimization

Extended duration of effect enables microRNA target analysis

Potent and long-lived tools to validate microRNA targets

miRIDIAN microRNA Mimic and Hairpin Inhibitors can serve as complementary molecular tools providing critical phenotypic information and rapid confi rmation of putative miRNA-target interactions. The combination of gain-of-function (mimic-induced down regulation) and loss-of function (inhibitor-induced up regulation) experiments provide the necessary support to demonstrate microRNA-target relationships.

miRIDIAN microRNA Mimics

miRIDIAN microRNA Hairpin Inhibitors

miRIDIAN microRNA Mimic and Hairpin Inhibitor Negative Controls

miRIDIAN microRNA Mimic and Hairpin Inhibitor Positive Controls

Modulation of AldoA mRNA levels in Huh-7 cell line using miRNA miRIDIAN Mimics & Inhibitors. Orange line indicates AldoA expression level as a result of endogenous miR-122 regulation. AldoA mRNA expression values >1 indicates inhibition of miR-122, while values < 1 indicates additional down regulation of AldoA by miRNA. The miRIDIAN microRNA Inhibitor resulted in an increase in Aldo A mRNA levels even at 7 days, whereas a standard 2'-O-methyl-RNA oligo complementary to mature miR-122 showed a small effect. The miRIDIAN microRNA Mimic immediately reduced Aldo A mRNA levels and sustained suppression throughout the experiment.

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miRIDIAN Hairpin Inhibitor: miR-122a

miRIDIAN Mimic: miR-122a

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Aldolase A is a putative target of miR-122 with a conserved 8-mer miR-122 predicted seed site in the 3' UTR. microRNA mimics and inhibitors designed to modulate endogenous miR-122 were transfected at several concentrations using DharmaFECT 1 into the human liver cell line, Huh-7 (10K cells), and assessed for their ability to alter Aldo A mRNA levels. Aldo A up- or down-regulation was determined using the Quantigene branched DNA assay (Panomics) at 1, 3, 5 and 7 days post-transfection.

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High-throughput Phenotypic Screening: miRIDIAN microRNA Mimic and Hairpin Inhibitor Libraries

Trust your screening results to the leader in RNAi technologies

miRIDIAN microRNA Mimic and Hairpin Inhibitor Libraries are complete collections of mimics and inhibitors in 96-well plates which enable classical genetic approaches to:

Why screen with miRIDIAN Libraries? Successful microRNA phenotypic screening requires best-in-class reagents to minimize false or misleading effects and achieve high-confi dence data. miRIDIAN Mimics and Hairpin Inhibitors are designed for robust and reproducible results.

Perform phenotypic high-throughput screening

Find potential biomarkers of normal or diseased cellular processes

Couple microRNA screening with high-content analysis for multi-parametric data

Identify microRNAs that synergize with drugs of interest for increased therapeutic benefi t

Mimics are modifi ed to prevent sense-strand uptake and ensure phenotypes resulting from only the mature microRNA

Hairpin inhibitors have long-lasting and potent inhibition, allowing enough time for full phenotypes to develop

Libraries are offered as complete collections for human, mouse and rat or as custom user-defi ned collections

PHENOTYPICEFFECTS

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Contact Information

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© 2009 Thermo Fisher Scientifi c Inc. All rights reserved. QuantiGene is a trademark of Bayer.

All other trademarks are the property of Thermo Fisher Scientifi c Inc. and its subsidiaries.

US Literature # 00074-06-D-03-U

Euro Literature # PB 2008 16

Thermo Scientifi c Dharmacon miRIDIAN

microRNA Mimics & Hairpin Inhibitors