New Tools for MicroRNA Functional Analysis
mirVana™ miRNA Mimics and Inhibitors
mirVana™ miRNA mimics and inhibitors provide a means to study the function of specific miRNAs in a range of organisms, and to validate their role in regulating target genes. We have released the second generation of these products, which are more specific (mimics) and potent (inhibitors) than their predecessors. mirVana™ oligonucleotides, available in both in vitro and in vivo formulations, reflect the most up-to-date Sanger miRBase content.
Mimics With Superior Specificity
mirVana™ miRNA mimics are small, chemically modified double-stranded RNA molecules that are designed to mimic endogenous mature miRNAs, resulting in artificial down-regulation of target mRNA translation and accompanied, in some cases, by reduction in transcript levels. Like natural miRNAs, these mimics have two strands—the mature strand, which is functional and used by the Argonaute (Ago) protein to target mRNAs; and the star strand, which is nonfunctional and is cleaved and expelled from the complex. The chemical modifications in mirVana™ miRNA mimics inactivate the star strand to help ensure that the guide strand (representing the desired mature miRNA) is taken up by Ago to produce the miRNA effect (Figure 1). mirVana™ miRNA mimics are designed to produce maximum and consistent effects at concentrations as low as 0.3 nM, and are available individually or as libraries.
Figure 1. High specificity of mirVana™ miRNA mimics. HeLa cells were cotransformed with one of six different miRNA mimics at 3 nM concentration and a corresponding reporter plasmid. The fold change in reporter gene expression for each miRNA mimic strand was determined by measuring expression in the presence of mimic relative to that of a negative control (set at 1.0). Each plasmid has the reporter gene cloned both in forward orientation (Fwd) to measure activity of the miRNA mimic mature strand, and in reverse/complement orientation (Rev) to test activity of the nonfunctional star strand. For all six mimics, mature strand activity is high (reporter gene expression reduced 5- to 10-fold compared with negative control), and star strand activity is low or absent (similar to negative control).
Inhibitors With Excellent Potency
mirVana™ miRNA inhibitors are chemically modified, single-stranded oligonucleotides designed to specifically bind to and inhibit endogenous miRNAs, resulting in artificial up-regulation of target mRNA translation. Compared with competitors, these inhibitors have the highest-potency in vitro inhibition at the lowest miRNA inhibitor concentration (Figure 2). Like the mimics, the mirVana™ miRNA inhibitors are available individually or as libraries.
Figure 2. mirVana™ miRNA inhibitors are more potent than products from leading competitors. mirVana™ miRNA inhibitors and two other commercially available let-7 inhibitors were each transfected into HeLa cells at 10 nM concentration using Lipofectamine RNAiMAX reagent. Twenty-four hours later, HMGA2 mRNA levels were measured by qRT-PCR using TaqMan assays (Roche Molecular Systems, Inc.).
Take Advantage of In Vivo–Ready Tools
mirVana™ miRNA mimics and inhibitors are compatible with both in vitro and in vivo applications; they have been validated with Lipofectamine RNAiMAX Transfection Reagent for use in cell-based systems (Figure 2), and with Invivofectamine 2.0 Reagent for in vivo delivery (Figures 3 and 4). These oligonucleotides are nontoxic and do not induce an immune response in the animal models tested. In vivo–ready mirVana™ miRNA mimics and inhibitors have been purified by HPLC and dialysis and are ready for immediate in vivo use.
Figure 3. mirVana™ miRNA inhibitors effectively suppress miRNA in vivo. miR-122 or Negative Control #1 mirVana™ miRNA inhibitors were complexed with Invivofectamine 2.0 reagent and injected into the tail veins of Balb-C mice on three consecutive days at 7 mg per kg body weight. Twenty-four hours after the last injection, expression levels of four natural targets of miR-122 were measured in the liver using TaqMan assays. Significant up-regulation of all four mRNAs was detected in mice treated with miR-122 inhibitor, as compared with mice that received no treatment or the negative control. Results indicate that mirVana™ miRNA inhibitors were efficiently delivered to the liver with Invivofectamine 2.0, where they inactivated miR-122, leading to up-regulation of genes naturally suppressed by miR-122.
|Figure 4. Fluorescently labeled mirVana™ miRNA mimcs and inhibitors enable tracking in vitro and in vivo. (A) Alexa Fluor 647 dye–labeled inhibitor was complexed with Invivofectamine 2.0 reagent and injected into the tail veins of Balb-C mice. Twenty-four hours later, animals were sacrificed and their livers sectioned and prepared for analysis. mirVana™ miRNA inhibitors are readily visualized in the liver section (red, Alexa Fluor 647 dye–labeled miRNA; green, Alexa Fluor 488 Phalloidin; blue, DAPI), indicating efficient uptake into hepatocytes. (B) Liver section from a mouse treated with unlabeled mirVana™ miRNA inhibitor. Fluorescently labeled mimic and inhibitors can be custom-ordered upon request; note that fluorescent labels do not affect the biological activity of mimics or inhibitors.|
Validated Positive and Negative Controls
The mirVana™ miRNA mimic miR-1 Positive Control is routinely used in experiments utilizing mirVana™ miRNA mimics. This mimic effectively down-regulates the expression of twinfilin-1, also known as PTK9, at the mRNA level (60–95% reduction). mirVana™ miRNA mimic Negative Control #1 has a unique sequence designed such that it does not target any human, mouse, or rat genes; it has been tested in human and mouse cell lines and tissues and validated to not produce identifiable effects on known miRNA functions.
The mirVana™ miRNA inhibitor let-7 Positive Control is validated for experiments using mirVana™ miRNA inhibitors. Endogenous let-7 miRNA negatively regulates HMGA2, a ubiquitously expressed nonhistone chromatin protein that modulates gene expression through changes in chromatin architecture. Upon introduction of the let-7 inhibitor, significant elevation in HMGA2 mRNA can be measured by qRT-PCR (Figure 2). The mirVana™ miRNA inhibitor Negative Control #1 has a unique sequence designed such that it does not target any human, mouse, or rat genes; it has been tested in human and mouse cell lines and tissues and validated to not produce any measurable effects on known miRNA functions.
Explore the Possibilities
For effective screening of multiple miRNAs, mirVana™ mimic and inhibitor libraries are available for miRNAs catalogued in version 17 of the miRBase sequence database. Explore our selection of mirVana™ miRNAs.