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Qiagen/miRCURY LNA miRNA Mimics/339173

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339173

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Functional & Cell Analysis
Gene Reporter Assays5
miRNA Functional Analysis5
RNA Silencing8
Transfection8

miRCURY LNA miRNA Mimics

For studies on miRNA function and gene regulation using synthetic miRNA

Third generation of highly potent, mature miRNA mimics with unique triple RNA strand design
No miRNA-star activity from bisected LNA-enhanced passenger strand
miRNA strand sequence matches miRBase annotation
Available with fluorescent label to assess transfection efficiency
Available with biotinylated miRNA strand to isolate targets by RNA pull-down

miRCURY LNA miRNA Mimicsare designed to simulate naturally occurring mature miRNAs.Introducing an miRNA mimic into cells increases the proportion of RNA-induced silencing complexes (RISC) containing the guide strand miRNA, enabling assessment of the phenotypic consequences of increased activity and discovery of newmiRNA functions. miRCURY LNA miRNA Mimics have an innovative design that includes two short,LNA-enhanced complimentary strands that prevent any miRNA-like activity associated with the passenger strands, so you can be sure that phenotypes observed using these mimics are due to increased activity of the mimicked miRNA.

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Cat No./ID:339173

miRCURY LNA miRNA Mimic (5 nmol)

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5 nmol miRCURY LNA miRNA Mimics with option of FAM label, provided in tubes

Cat No./ID:339174

miRCURY LNA miRNA Mimic (20 nmol)

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20 nmol miRCURY LNA miRNA Mimics with option of FAM label, provided in tubes

Cat No./ID:339178

miRCURY LNA Premium miRNA Mimic (5 nmol)

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5 nmol miRCURY LNA Premium miRNA Mimics, HPLC purified, with biotin label, provided in tubes

Cat No./ID:339179

miRCURY LNA Premium miRNA Mimic (20 nmol)

Go to GeneGlobe

20 nmol miRCURY LNA Premium miRNA Mimics, HPLC purified, with biotin label, provided in tubes

miRCURY LNA miRNA Mimics are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

miRNA target identification with biotinylated mimics.

Biotinylated miRNA mimics are delivered to cells by transfection. Cells are harvested and lysed 24 H later. Complexes of biotinylated RISC and mRNA are pulled down with streptavidin-coated magnetic beads. After washing, the captured RNA is purified and analyzed with microarrays, RT-qPCR or by next-generation sequencing.

The distinct RNA triplex design ensures completely specific miRNA mimicry.

Only the miRNA strand is incorporated by RISC. The two passenger strands are too short to act as miRNAs and are rapidly degraded after displacement from the miRNA strand. Off-target effects from the passenger strands are therefore minimal with miRCURY LNA miRNA Mimics.

Biotinylated miRCURY LNA miRNA Mimics.

Biotinylated miRCURY LNA miRNA Mimics consist of three RNA strands: a3’ biotinylated miRNA (guide) strand with sequence exactly according to miRBase annotation and the passenger strand, whichis split into two LNA-modified RNA strands complementary to the miRNA strand. Only the miRNA strand is incorporated by the RISC. The two passenger strands are too short to act as miRNAs and are rapidly degraded after displacement from the miRNA strand.

Fluorescently labeled miRNA mimics.

Note that the fluorophore is attached to the 5’ passenger strand and not to the miRNA guide strand.

Silencing of miRNA reporter assays with fluorescently labeled miRCURY LNA miRNA Mimics.

Introduction of fluorophore at the 5" end of the passenger strand has no adverse effects on the miRNA acitivity of the mimic. HeLa cells harboring an hsa-miR-1-3p luciferase reporter plasmid were transfected with fluorescence-labeled and non-labeled hsa-miR-1-3p mimics and Cel-miR-39-3p negative mimic control. The results demonstrate that suppression of luciferase actity is achieved equally efficiently with fluorescence-labeled and non-labeled mimics.

Perfect miRNA strand-specific activity with miRCURY LNA miRNA Mimics.

HeLa cells harboring hsa-miR-16-3p (top panel) and hsa-miR-16-5p (bottom panel) luciferase reporter plasmids,were transfected with hsa-miR-16-3p and hsa-miR-16-5p mimics, respectively,and Cel-miR-39-3p negative mimic control. The results demonstrate that suppression of luciferase actity is only achieved with the miRCURY LNA miRNA Mimic corresponding to the reporter assay.

Silencing of miRNA reporter assays with biotinylated miRCURY LNA miRNA Mimics.

HeLa cells were transfected with a plasmid harboring a Renilla luciferase gene (transfection efficiency control) and a firefly luciferase reporter gene with a sequence complementary to hsa-miR- 1-3p in the 3" UTR (pmiR-1). The next day, the cell cultures were transfected with different concentrations of hsa-miR-1-3p miRNA mimic or Cel-miR-39-3p negative control. Reporter gene expression was measured 24H after transfection with a dual luciferase assay. Ratios of firefly and Renilla luciferase activity were calculated and normalized to values obtained with a firefly luciferase reporter with no miR target sequence (pLuc). The results illustrate that potent miRNA activity can be achieved with biotinylated mimics even if they are ~50x less active than unlabeled mimics.

A unique triple strand design

miRCURY LNA miRNA Mimics consist of three RNA strands. An unmodified miRNA (guide) strand has a sequence exactly matching the miRBase annotation. The passenger strand is split into two LNA-modified RNA strands complementary to the miRNA strand.

Silencing of miRNA reporter assays with miRCURY LNA miRNA Mimics.

HeLa cells were transfected with a plasmid with a Renilla luciferase (transfection efficiency control) and miRNA target sequence in the 3’UTR of a firefly luciferase reporter gene. The nextday, the cell cultures were transfected with different concentrations of the corresponding miRCURY LNA miRNA Mimics or cel-miR-39-3p negative control. Firefly luciferase expression is suppressed by the corresponding mimic at endogenous miRNA levels in the cell. Reporter gene expression was measured 24H after transfection with a dual luciferase assay. Ratios of firefly and Renilla luciferase activity were calculated and normalized to values obtained with a firefly luciferase reporter with no miR target sequence (pLuc). The results illustrate that our mimics display sub-nanomolar potency under optimal transfection conditions.

Performance

Excellent potency

The miRNA strand of our mimics is made of unmodified RNA to provide as accurate miRNA mimicry as possible. The sequence and LNA spiking pattern of the two complementary passenger strands have been carefully designed to optimize efficient incorporation of the miRNA (guide) strand into RISC (see figureSilencing of miRNA reporters using mimics for examples of the high potency of these LNA mimics).

No miRNA star-like activity from the passenger strand

miRCURY LNA miRNA Mimics have an innovative design, which includes two short LNA-enhanced complimentary strands that prevent any miRNA-like activity associated with the passenger strands (see figure The distinct RNA triplex design ensures completely specific miRNA mimicry). This guarantees that phenotypes observed using these mimics are due to increased activity of the mimicked miRNA.

Fluorescently labeled miRNA mimics

Fluorescently labeled miRNA mimics can be useful to assess transfection efficiency. However, addition of fluorescent labels to the 5’ or 3’ end of the miRNA strand completely removes activity of miRNA mimics. In contrast, fluorophores at the 5’ end of the passenger strand are tolerated and have no adverse effects on the resulting mRNA activity (see figuresFluorescently labeled miRNA mimics and Biotinylated miRCURY LNA miRNA mimics). Because the fluorophore is attached to the passenger strand, the fluorescent signal does not report on the intracellular localization of the miRNA strand that is loaded into RISC and is only useful for monitoring transfection efficiency (see figure Silencing of miRNA reporter assays with fluorescently labeled miRCURY LNA miRNA Mimics).

Biotinylated miRNA mimics

Modification of the 5’ and 3’ ends of the miRNA strand in mimics is generally not well tolerated at all. However, we have observed that potent miRNA activity is achievable with biotinylated mimics, although they are significantly less potent than non-modified mimics, presumably due to less efficient loading into RISC. Nevertheless, the possibility that biotin modification of the guide strand creates a bias for incorporation of the passenger strand cannot be excluded. It is therefore important to use modified mimics that ensure that only the miRNA strand is chosen by RISC. The special triple RNA strand design of miRCURY LNA miRNA mimics ensures complete miRNA strand specificity, since the passenger strand is split into two LNA-modified RNA strands (see figuresThe distinct RNA triplex design ensures completely specific miRNA mimicry and Silencing of miRNA reporter assays with biotinylated miRCURY LNA miRNA Mimics).

Principle

miRCURY LNA miRNA Mimics have a unique and innovative design that includesthree RNA strands, rather than the two RNA strands that characterize traditional miRNA mimics. The miRNA (guide) strand is an unmodified RNA strand with a sequence corresponding exactly to the annotation in miRBase. However, the passenger strand is divided into two LNA-enhanced RNA strands (see figureA unique triple RNA strand design). When designed correctly, these triple RNA strand mimics are as potent as traditional double-strand RNA mimics. The great advantage is that the segmented nature of the passenger strand ensures that only the miRNA strand is loaded into the RNA-induced silencing complex (RISC) with no resulting miRNA activity from the two complementary passenger strands. Phenotypic changes observed with miRCURY LNA miRNA mimics can therefore be safely ascribed to the miRNA simulated by the mimic (see figuremiRNA target identification with biotinylated mimics).The distinct triple RNA strand design is enabled by incorporation of high-affinityLNA nucleotides into the two passenger strands. The sequence, length and LNA spiking pattern of the two passenger strands have been optimized using a sophisticated and empirically derived design algorithm. miRCURY LNA miRNA Mimics have been predesigned for a large majority of human, mouse and rat miRNAs listed in miRBase. Since many miRNAs are phylogenetically conserved, this set of miRNA mimics covers a large proportion of vertebrate and invertebrate miRNAs.

Procedure

Following resuspension, miCURY LNA miRNA Mimics are transfected into cells with a transfection reagent or via electroporation. Phenotypic effects of the miRNA mimic are normally assessed 24–72 hours after transfection. For some applications, such as cell differentiation assays, the phenotypic readout may take place 7–10 days after transfection.

Applications

miRNA mimicssimulate the natural functions of endogenous miRNAs and are primarily used in gain-of-function studies by assessing the biological consequences of increasing miRNA activity.The effect of increasing the cellular content of an miRNA using miRNA mimics can be studied in numerous ways, including cellular assays to monitor cell proliferation, cell differentiation or apoptosis. The effect on gene expression can also be measured at the mRNA or protein level of putative miRNA targets.miRNA mimics are also frequently used for validating miRNA targets in combination with miRNA inhibitors and target site blockers. Typically, plasmid-based assays are used in which the 3" UTR of the mRNA under investigation has been cloned downstream of a reporter gene. Introducing the mimic into cells harboring the reporter plasmid will reduce reporter gene expression, while miRNA inhibitors and target site blockers masking the 3" UTR miRNA binding site will cause derepression.Biotinylated miRNA mimics are highly effective tools for identification of miRNA targets in RNA pull-down experiments. Recent advances with this experimental approach are transforming our understanding of miRNA biology and have revealed that non-canonical miRNA–mRNA interactions, which are ignored by target prediction tools, are frequent and lead to target repression. The principle of the technique is shown in Perfect miRNA strand-specific activity with miRNA mimics.Biotinylated mimics are significantly less potent than non-modified mimics.

Product Resources

Brochures & Guides (1)

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	RNA Functional Analysis – enhanced by LNA	Show details

Kit Handbooks (1)

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	miRCURY LNA miRNA Mimics Handbook	Show details

Scientific Posters (1)

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	Explore the RNA Universe! Poster for download	Show details

Safety Data Sheets (4)

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	MSDS miRCURY LNA miRNA Mimic (5 nmol)
	MSDS miRCURY LNA miRNA Mimic (20 nmol)
	MSDS miRCURY LNA Premium miRNA Mimic (5 nmol)
	MSDS miRCURY LNA Premium miRNA Mimic (20 nmol)

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