miScript Target Protectors

For miRNA functional studies performed by interfering with miRNA-target interaction

  • An innovative tool for miRNA discovery
  • Ready-to-transfect in miRNA regulation experiments
  • Specific protection of a single target for precise functional studies
  • Custom design for protection of human, mouse, or rat targets
miScript Target Protectors are single-stranded, modified RNAs that specifically interfere with the interaction of an miRNA with a single target, while leaving the regulation of other targets of the same miRNA unaffected. Transfection of miScript Target Protectors, followed by analysis of changes in phenotype or gene expression, can enable elucidation of the targets and roles of particular miRNAs.
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miScript Target Protector
5 nmol cell-culture–grade target protector
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miScript Target Protectors are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Reliable miRNA inhibition.|Target verification.|miScript Target Protectors are specific for the gene of interest.|Inhibition of miRNA regulation of BCL-2.|Phenotype changes after miScript Target Protector transfection.|
miR-16 is endogenously expressed in HeLa S3 and MCF-7 cells. A luciferase reporter construct with a binding site for miR-16 was cotransfected with various amounts of a miScript Target Protector for the same binding site. All amounts resulted in significant increase in luciferase expression caused by inhibition of miRNA downregulation. Neg. control: Luciferase construct cotransfected with Negative Control miScript Target Protector. Unregulated: Luciferase construct without the miR-16 binding site. Attractene Transfection Reagent was used for cotransfections.|MCF-7 cells were cotransfected with miR-29a miScript miRNA Mimic (10 nM) plus Negative Control miScript Target Protector (Mimic) or miR-29a miScript miRNA Mimic plus a miScript Target Protector (1 µM) specific for the putative miR-29a binding site of DNMT3A (Mimic + target protector). Transfections were performed using HiPerFect Transfection Reagent. Forty-eight hours after transfection, expression analysis of DNMT3A was performed by real-time RT-PCR. Untransfected cells were also analyzed. Negative control: Transfection of AllStars Negative Control siRNA and Negative Control miScript Target Protector.|miR-1 downregulates expression of ADAR and HDAC4 genes. HeLa cells were cotransfected with a miScript Target Protector for the miR-1 binding site of ADAR (1 µM, 0.5 µM, 0.1 µM, or 0.05 µM) and miR-1 miScript miRNA Mimic (10 nM). Transfections were performed using HiPerFect Transfection Reagent. After 72 hours, ADAR expression [A] and HDAC4 expression [B] was detected by western blotting. Tubulin was detected as an internal control. Untransfected cells were also analyzed (UT). Negative control (Neg.): Cotransfection of Negative Control miScript Target Protector and miR-1 miScript miRNA Mimic. The miScript Target Protector for the miR-1 binding site of ADAR specifically protected ADAR from miR-1 mediated downregulation and did not affect downregulation of HDAC4 by miR-1.|MCF-7 cells were cotransfected with a miScript Target Protector for the miR-15a/miR-16 binding site of BCL-2 (500 nM or 1 µM) and miR-16 miScript miRNA Mimic (10 nM). After 72 hours, BCL-2 expression was detected by western blotting. Cells transfected with mimic and target protector showed an increase in expression compared to the negative control. Negative control (Neg.): Cotransfection of miR-16 miScript miRNA Mimic and Negative Control miScript Target Protector. Tubulin was also detected as an internal control. Transfections were performed using HiPerFect Transfection Reagent.|MCF-7 cells were transfected with 10 nM miR-9 miScript miRNA Mimic or cotransfected with 10 nM miR-9 miScript miRNA Mimic plus 1 µM miScript Target Protector for the miR-9 binding site of the cell viability gene. After 72 hours, cell viability was examined by light microscopy. Transfections were performed using HiPerFect Transfection Reagent.|
Performance
Specific inhibition of miRNA regulation of a single target

The design and modification of miScript Target Protectors ensures specific, efficient binding to the miRNA-binding site of interest. Experiments with a luciferase reporter construct showed reliable, reproducible miRNA inhibition after transfection of miScript Target Protector (see figure "Reliable miRNA inhibition"). miScript Target Protectors were shown to protect the miRNA-binding site of the target gene, while leaving other gene targets of the same miRNA unaffected (see figure "miScript Target Protectors are specific for the gene of interest").

Reliable verification of miRNA-targeted genes

miScript Target Protectors can be used to provide evidence that a gene is regulated by a particular miRNA. The DNMT3A gene encodes DNA methyltransferase 3, which is involved in development and differentiation. TargetScan predicted a single binding site for miR-29a in the 3' UTR of DNMT3A. Transfection of a miR-29a miScript miRNA Mimic led to downregulation of DNMT3A as shown by real-time RT-PCR, indicating that miR-29a downregulates DNMT3A expression at the transcriptional level. Transfection of a miScript Target Protector designed for the miR-29a binding site predicted by TargetScan resulted in increase in DNMT3A expression. These experiments provided convincing evidence that DNMT3A is negatively regulated by miR-29a via the binding site predicted by TargetScan (see figure "Target verification").

miRNA regulation identified at protein level

Gene expression can be downregulated by miRNA at the transcriptional level or at the protein level. miScript Target Protectors inhibit miRNA regulation at both levels. Human BCL-2 is known to be regulated by 2 endogenously expressed miRNAs: miR-15a and miR-16. Both miR-15a and miR-16 use the same binding site in the 3' UTR of BCL-2. Transfection of a miScript Target Protector for this binding site resulted in increase in BCL-2 expression at the protein level (see figure "Inhibition of miRNA regulation of BCL-2").

miRNA regulation affects phenotype

Effects on a cellular process or pathway can result in phenotypic changes such as alterations in cell morphology or cell viability. Observation of a change in phenotype after transfection of a miScript Target Protector indicates that the miRNA and target of interest play a role in the pathway/process. miR-9 causes downregulation of a gene that is important for cell viability. In MCF-7 cells, transfection of miR-9 miScript miRNA Mimic resulted in high levels of cell death. However, cotransfection of miR-9 miScript miRNA Mimic and a miScript Target Protector designed for the mir-9 binding site of the gene overcame the cell death phenotype. This result indicated that the miScript Target Protector inhibited miR-9 downregulation allowing the cell viability gene to be expressed (see figure "Phenotype changes after miScript Target Protector transfection").

Principle

miScript Target Protectors are innovative tools that provide valuable insights into the roles of miRNAs in regulation of individual genes. A miScript Target Protector is designed to protect the miRNA-binding site of a specific target gene. After transfection, the miScript Target Protector binds to the miRNA-binding site, blocking miRNA access to the site and preventing gene downregulation by a specific miRNA.

Procedure
miScript Target Protector transfection is followed by phenotype or gene expression analysis. Increased target-gene expression, a change in signaling patterns, or an altered phenotype, can all provide evidence that the miRNA and target under study are involved in the pathway or phenotype of interest. The role of miRNAs in various pathways can be studied by examination of specific phenotypes after transfection of different miScript Target Protectors.

QIAGEN products for miRNA functional analysis also include miScript miRNA Mimics, which are synthetic miRNAs, and miScript miRNA Inhibitors, which inhibit the action of a particular miRNA on all its targets. miScript miRNA Mimics and Inhibitors can be used along with miScript Target Protectors to enable insightful and revealing discoveries of miRNA function.

Positive and negative controls

Positive and Negative Control miScript Target Protectors are also available to facilitate experimental setup and interpretation of results. The Negative Control miScript Target Protector has no homology to any known mammalian gene. The Positive Control miScript Target Protector is designed to protect endogenously expressed BCL-2 from miR-15a and miR-16. Transfection of the Positive Control miScript Target Protector provides a positive control that determines whether the experimental setup is working correctly

Applications

miScript Target Protectors, together with miScript miRNA Mimics and Inhibitors, can be used for a wide variety of miRNA functional studies, including:

Verification of miRNA binding sites
Determination of miRNA role in a pathway or process
Study of regulation of a gene of interest
Confirmation of results from experiments, such as microarray analysis
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Feature
Specifications
Design Customer design
Format Single tubes
Scale or yield 5 nmol
Sequence provided No
Species Human, mouse, rat

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