miRCURY LNA miRNA Inhibitor Libraries
For genomewide, high-throughput screening of miRNA function using LNA-enhanced miRNA inhibitors
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.
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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).
FeaturesmiRCURY 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.
DesignThis 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.
CoverageWe 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.
Plate layoutmiRCURY 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.
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.
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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