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miRCURY LNA miRNA Inhibitor Libraries

For genomewide, high-throughput screening of miRNA function using LNA-enhanced miRNA inhibitors
  • Uniform, high potency miRNA inhibition, regardless of GC content
  • Low toxicity and minimal secondary effects allow efficient miRNA targeting at low concentrations
  • High miRBase v.20 coverage: 1,972 human inhibitors or 1,624 mouse inhibitors
  • Excludes miRNAs with little experimental evidence for cost efficiency and smarter screening workflows
  • Practical 96-well format for high-throughput screening

miRCURY LNA miRNA Inhibitor Libraries enable high-throughput, genomewide screening of miRNA function. LNA enhancement makes it possible to normalize the inhibitors' Tms, ensuring highly efficient targeting of all miRNAs, regardless of GC content.

Please contact us for ordering and for further information, or email our Support Team directly.

Cat No./ID: 339165
miRCURY LNA miRNA Family Power Inhibitor Library (0.125 nmol)
0.125 nmol miRCURY LNA miRNA Family Power Inhibitor Libraries, provided in 96-well plate format
Cat No./ID: 339166
miRCURY LNA miRNA Family Power Inhibitor Library (0.25 nmol)
0.25 nmol miRCURY LNA miRNA Family Power Inhibitor Libraries, provided in 96-well plate format
Cat No./ID: 339168
miRCURY LNA miRNA Inhibitor Library (0.125 nmol)
0.125 nmol miRCURY LNA miRNA Inhibitor Libraries, provided in 96-well plate format
Cat No./ID: 339169
miRCURY LNA miRNA Inhibitor Library (0.25 nmol)
0.25 nmol miRCURY LNA miRNA Inhibitor Libraries, provided in 96-well plate format

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


0
Example of a miRCURY LNA miRNA Inhibitor Library plate.
This plate shows the layout of panel 10 of the human miRNA inhibitor library.
1
Examples of miRNA silencing with miRCURY LNA miRNA inhibitors.
HeLa cells were transfected with a plasmid containing Renilla luciferase for the transfection efficiency control and the miRNA target sequence of a firefly luciferase reporter gene in the 3’ UTR. Firefly luciferase expression is suppressed by the corresponding endogenous miRNA level in the cell. One day later, the cell cultures were transfected with various concentrations of the corresponding regular miRCURY LNA miRNA Inhibitor. Reporter gene expression was measured with a dual luciferase assay 24 H after transfection. 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 regular miRCURY LNA miRNA Inhibitors display sub-nanomolar potency under optimal transfection conditions.
2
miRNA silencing via direct uptake (gymnosis) of Power Inhibitors.
HepG2, HeLa and HEK293 cells were transfected with a plasmid encoding Renilla luciferase (transfection efficiency control) and a firefly luciferase reporter gene with either an miR-21-5p target site or an miR-27a-3p target site in the 3’-UTR (pmiR-21-5p or pmiR-27a-3p). 24 H after removal of the transfection reagent, corresponding miRCURY LNA miRNA Power Inhibitors were added directly to culture medium in different concentrations. Reporter gene expression was measured with a dual luciferase assay 48 H (HepG2) and 72 H (HeLa and HEK293) after addition of the inhibitors. 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) in each of the three cell lines.

The results illustrate that efficient miRNA inhibition can be achieved by adding high concentrations of Power Inhibitor directly to the culture medium. However, uptake kinetics with gymnosis is slower than delivery of the inhibitors using transfection reagents. When using transfection reagents, we normally observe strong inhibition after just 24 H. With unassisted uptake, we observe activity with some cell lines and certain inhibitors after one day, but it typically peaks between 48–72 H after addition of the inhibitors. Normal inhibitors with an unmodified, normal phosphodiester backbone are ineffective with gymnotic delivery, probably due to insufficient stability.

Performance
Tm-normalized miRCURY LNA miRNA Inhibitors have unmatched potency against all miRNAs, regardless of their GC content (see figure Examples of miRNA silencing with miRCURY LNA miRNA inhibitors). The Power Inhibitors are so potent that they can be added directly to cell cultures without the need for transfection reagents (see figure miRNA silencing via direct uptake (gymnosis) of Power Inhibitors).
Principle
Features
miRCURY LNA miRNA Inhibitor Libraries allow convenient high-throughput screening of mouse and human miRNA function. These libraries enable the discovery of the collective function of miRNA families that might otherwise go unnoticed using our normal inhibitors that address individual (and perhaps redundant) miRNA family members. The miRCURY LNA miRNA Inhibitor Libraries are based on our renowned Tm-normalized miRCURY LNA miRNA Inhibitors with phosphodiester backbones.

Design
This third generation of miRCURY LNA miRNA Inhibitor Libraries contain inhibitors that have been completely redesigned using an improved LNA design algorithm. By intelligent LNA spiking, we have developed inhibitors that all have a Tm within a narrow range around an empirically determined optimal value, regardless of their GC content. In addition, the level of self-complementarity has been kept to an insignificant, low level.

The LNA spiking pattern also ensures increased resistance to enzymatic degradation and long-lasting biological effects. As a result, these inhibitors feature a high, uniform potency towards all miRNA targets. This is of great value if you are conducting large screening projects using uniform transfection conditions for all inhibitors in the library.

In addition to high specificity of these inhibitors for their target miRNAs, the carefully designed LNA spiking pattern ensures that potential mRNA–inhibitor duplexes are not recognized as RNase H targets. This minimizes potential off-target effects and reduces the risk that any observed biological phenotype is caused by factors other than the antisense inhibition of the miRNA target.

Coverage
We offer a high coverage of miRNAs listed in miRBase v.20. However, we have intentionally excluded a number of miRNAs for which there is no or very limited direct experimental evidence. This significantly reduces the cost of screening and time wasted on potentially false-positive results, and does not impact the true coverage of the screen.
  • Human miRNA inhibitor library: 1,972 inhibitors of human miRNAs listed in miRBase v. 20, corresponding to a coverage of 78%
  • Mouse miRNA inhibitor library: 1,624 inhibitors of mouse miRNAs listed in miRBase v. 20, corresponding to a coverage of 86%
  • miRNA Family inhibitor library: a collection of all 43 of our miRCURY LNA miRNA Power Family Inhibitors. The Power Inhibitors address miRNA families conserved in human and mouse.

Plate layout
miRCURY LNA miRNA Inhibitor Libraries are provided in 96-well plates, which are are all organized as shown in the figure Example of a miRCURY LNA miRNA Inhibitor Library plate. The empty outer rows and columns facilitate easy pipetting into 96-well culture plates in a setup that avoids edge effects from evaporation of culture medium.

Well B2 is left empty for a control oligonucleotide of your choice. For example, this could be one of our negative controls with or without a FAM label for visual inspection of transfection efficiency. This oligonucleotide must be purchased separately and manually added to well B2 in the plates.

A positive transfection control is provided in well B3. This control is a toxic oligonucleotide, and efficient transfection with this oligonucleotide will cause cell death.

We have generated an miRNA ranking based on factors such as the number of publications, type of experimental evidence, number of sequencing experiments and number of reads. We use this ranking to position the inhibitors in the plates in a descending order. The inhibitors with the highest score (the best characterized) are positioned in the first plate, and the inhibitors with the lowest score are positioned in the last plate. Organizing the inhibitors in the plates according to the amount of supporting scientific data enables smarter screening workflows with a subset of the plates containing inhibitors of the best-validated miRNAs without the need for laborious pipetting and reformatting of the library.
Procedure
miRCURY LNA miRNA Inhibitors are antisense oligonucleotides with perfect sequence complementary to their targets. When introduced into cells, they sequester the target miRNA in highly stable heteroduplexes, effectively preventing the miRNA from hybridizing with its normal cellular interaction partners. The sequences of the oligonucleotides and their LNA spiking patterns have been carefully designed to achieve uniform high potency for all miRCURY LNA miRNA Inhibitors, regardless of the GC-content of the target. The Tm is normalized to an optimal temperature, and the level of self-complementarity is kept to a minimum.

Following resuspension, miCURY LNA miRNA Inhibitors are transfected into cells with a transfection reagent or via electroporation. Phenotypic effects of the miRNA inhibitor 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
miRCURY LNA miRNA Inhibitors are primarily used miRNA functional studies by assessing the biological consequences of inhibiting miRNA activity. These effects can be assessed in a variety of ways, including using cellular assays to monitor cell proliferation, cell differentiation or apoptosis. The effects on gene expression can also be measured at the mRNA or protein level of putative miRNA targets.
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