Workflow features

  • Gain- and loss-of-function phenotypes identify genes and processes regulated by miRNAs
  • Western blotting and/or real-time RT-PCR are used to validate miRNA targets
  • Target site interaction and impact on protein expression can be evaluated using reporter gene and immunodetection systems

See Literature/References

How it works
  • Ambion® Pre-miR™ miRNA Precursors and Anti-miR™ miRNA Inhibitors are designed to mimic or inhibit specific miRNAs for artificial upregulation and downregulation of target mRNA translation, respectively
  • miRNA targets are validated by quantitating target protein and/or messenger RNA levels in response to miRNA upregulation or downregulation
  • Western analysis is used to investigate the impact on protein expression
  • Reporter gene assays are used to study the interaction between miRNAs and their target sites
miRNA functional analysis experimental workflow

Ambion® Pre-miR™ miRNA Precursors and Anti-miR™ miRNA Inhibitors are designed to mimic or inhibit specific miRNAs for artificial upregulation and downregulation of target mRNA translation, respectively.

Recommended Products

 

Anti-miR™ miRNA Inhibitors
Ambion® Anti-miR™ miRNA Inhibitors are chemically modified, single stranded nucleic acids designed to specifically bind to and inhibit endogenous miRNA molecules. These ready-to-use inhibitors can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs, and enable detailed study of miRNA biological effects.

 

Pre-miR™ miRNA Precursors
Ambion® Pre-miR™ miRNA Precursor Molecules are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNA molecules. These ready-to-use miRNA mimics, which are similar to but not identical to siRNAs, can be introduced into cells using transfection or electroporation parameters identical to those used for siRNAs and enable detailed study of miRNA biological effects via gain-of-function experiments.

The siPORT™ NeoFX™ Transfection Agent efficiently and reproducibly delivers miRNA precursors and inhibitors into a broad range of mammalian cell types with minimal toxicity.

Recommended Products

 

Pre-miR™ miRNA Starter Kit
The Pre-miR™ miRNA Starter Kit offers a complete solution for controlling and optimizing miRNA gain-of-function experiments in human cells mediated by synthetic miRNA mimics. The kit includes positive and negative control Pre-miR™ miRNA Precursors, a matched real-time PCR assay, and a transfection agent.

 

siPORT™ NeoFX™ Transfection Agent
siPORT™ NeoFX™’s lipid-based formulation enables the transfection of siRNA into adherent cells as they are subcultured — without increased cytotoxicity. From start to finish, successful gene silencing experiments can be completed in as little as 24 hours. This streamlined protocol can be adapted to a wide range of cells and experimental designs, including high-throughput applications.

The TaqMan® Gene Expression Cells-to-CT™ Kit enables researchers to easily perform expression analysis using cultured cells without purification of RNA

Recommended Products

 

TaqMan® Gene Expression Cells-to-CT™ Kit
The kit includes reagents for cell lysis and RNA preservation, genomic DNA removal, and reverse transcription (RT). The kit also includes TaqMan® Gene Expression Master Mix for use in subsequent real-time PCR analysis.

 

TaqMan® Gene Expression Assays
Applied Biosystems offers more than 1.2 million TaqMan® Gene Expression Assays, the most comprehensive set of predesigned real-time PCR assays available. All TaqMan® Gene Expression Assays have been designed using our validated bioinformatics pipeline, and run with the same PCR protocol, eliminating the need for primer design or PCR optimization.

 

StepOnePlus™ Real-Time PCR System
The Applied Biosystems® StepOnePlus™ Real-Time PCR System makes it simple and easy to get publication-ready high-quality real-time PCR results on day one. This remarkably simple 96-well, 4-color, real-time PCR system is designed with a user-friendly, yet powerful, interface for researchers of all experience levels.

 

7500 & 7500 Fast Real-Time PCR Systems
The Applied Biosystems® 7500 Fast Real-Time PCR System offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast System delivers high-quality results in as little as 30 minutes.

The Applied Biosystems® 7500 Fast Real-Time PCR System offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast System delivers high-quality results in as little as 30 minutes.

 

7900HT Fast Real-Time PCR System
The 7900HT Fast Real-Time PCR System combines 384-well plate compatibility with fully automated robotic loading — and also offers optional Fast real-time PCR capability. Acknowledged as the gold standard in real-time PCR, the 7900HT System combined with TaqMan® Genomic Assays enables you to achieve unprecedented throughput and flexibility, allowing you to pursue projects beyond the scope of previous real-time instruments.

miRNA functional studies may require simultaneous analyses of RNA and protein expression. The Western-SuperStar™ Immunodetection System is a highly sensitive chemiluminescent immunodetection system, providing an ideal solution for measuring the effect of miRNA function on the expression of specific proteins.

Recommended Products

 

Western-SuperStar™ Immunodetection System
Western-SuperStar™ Immunodetection System is a highly sensitive, convenient chemiluminescent immunodetection system. The Western-SuperStar  System is ideal for confirmation of protein knockdown in siRNA-mediated gene silencing experiments.

 

pMIR-REPORT™ miRNA Expression Reporter Vector System
The pMIR-REPORT™ miRNA Expression Reporter Vector System provides accurate, quantitative, in-cell measurement of miRNA expression.

 

Dual-Light® Combined Reporter Gene Assay System
The Dual-Light® luminescent reporter gene assay is designed for the rapid and sensitive detection of firefly luciferase and b-galactosidase in a single extract aliquot for gene expression assays.

Step 1 : Select miRNA Mimics or Inhibitors

Ambion® Pre-miR™ miRNA Precursors and Anti-miR™ miRNA Inhibitors are designed to mimic or inhibit specific miRNAs for artificial upregulation and downregulation of target mRNA translation, respectively.

Recommended Products

 

Anti-miR™ miRNA Inhibitors
Ambion® Anti-miR™ miRNA Inhibitors are chemically modified, single stranded nucleic acids designed to specifically bind to and inhibit endogenous miRNA molecules. These ready-to-use inhibitors can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs, and enable detailed study of miRNA biological effects.

 

Pre-miR™ miRNA Precursors
Ambion® Pre-miR™ miRNA Precursor Molecules are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNA molecules. These ready-to-use miRNA mimics, which are similar to but not identical to siRNAs, can be introduced into cells using transfection or electroporation parameters identical to those used for siRNAs and enable detailed study of miRNA biological effects via gain-of-function experiments.

Step 2 : Transfect Cells

The siPORT™ NeoFX™ Transfection Agent efficiently and reproducibly delivers miRNA precursors and inhibitors into a broad range of mammalian cell types with minimal toxicity.

Recommended Products

 

Pre-miR™ miRNA Starter Kit
The Pre-miR™ miRNA Starter Kit offers a complete solution for controlling and optimizing miRNA gain-of-function experiments in human cells mediated by synthetic miRNA mimics. The kit includes positive and negative control Pre-miR™ miRNA Precursors, a matched real-time PCR assay, and a transfection agent.

 

siPORT™ NeoFX™ Transfection Agent
siPORT™ NeoFX™’s lipid-based formulation enables the transfection of siRNA into adherent cells as they are subcultured — without increased cytotoxicity. From start to finish, successful gene silencing experiments can be completed in as little as 24 hours. This streamlined protocol can be adapted to a wide range of cells and experimental designs, including high-throughput applications.

Step 3 : Assess Target mRNA Levels (optional)

The TaqMan® Gene Expression Cells-to-CT™ Kit enables researchers to easily perform expression analysis using cultured cells without purification of RNA

Recommended Products

 

TaqMan® Gene Expression Cells-to-CT™ Kit
The kit includes reagents for cell lysis and RNA preservation, genomic DNA removal, and reverse transcription (RT). The kit also includes TaqMan® Gene Expression Master Mix for use in subsequent real-time PCR analysis.

 

TaqMan® Gene Expression Assays
Applied Biosystems offers more than 1.2 million TaqMan® Gene Expression Assays, the most comprehensive set of predesigned real-time PCR assays available. All TaqMan® Gene Expression Assays have been designed using our validated bioinformatics pipeline, and run with the same PCR protocol, eliminating the need for primer design or PCR optimization.

 

StepOnePlus™ Real-Time PCR System
The Applied Biosystems® StepOnePlus™ Real-Time PCR System makes it simple and easy to get publication-ready high-quality real-time PCR results on day one. This remarkably simple 96-well, 4-color, real-time PCR system is designed with a user-friendly, yet powerful, interface for researchers of all experience levels.

 

7500 & 7500 Fast Real-Time PCR Systems
The Applied Biosystems® 7500 Fast Real-Time PCR System offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast System delivers high-quality results in as little as 30 minutes.

The Applied Biosystems® 7500 Fast Real-Time PCR System offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast System delivers high-quality results in as little as 30 minutes.

 

7900HT Fast Real-Time PCR System
The 7900HT Fast Real-Time PCR System combines 384-well plate compatibility with fully automated robotic loading — and also offers optional Fast real-time PCR capability. Acknowledged as the gold standard in real-time PCR, the 7900HT System combined with TaqMan® Genomic Assays enables you to achieve unprecedented throughput and flexibility, allowing you to pursue projects beyond the scope of previous real-time instruments.

Step 4 : Assess Target Protein Levels/Other Effects

miRNA functional studies may require simultaneous analyses of RNA and protein expression. The Western-SuperStar™ Immunodetection System is a highly sensitive chemiluminescent immunodetection system, providing an ideal solution for measuring the effect of miRNA function on the expression of specific proteins.

Recommended Products

 

Western-SuperStar™ Immunodetection System
Western-SuperStar™ Immunodetection System is a highly sensitive, convenient chemiluminescent immunodetection system. The Western-SuperStar  System is ideal for confirmation of protein knockdown in siRNA-mediated gene silencing experiments.

 

pMIR-REPORT™ miRNA Expression Reporter Vector System
The pMIR-REPORT™ miRNA Expression Reporter Vector System provides accurate, quantitative, in-cell measurement of miRNA expression.

 

Dual-Light® Combined Reporter Gene Assay System
The Dual-Light® luminescent reporter gene assay is designed for the rapid and sensitive detection of firefly luciferase and b-galactosidase in a single extract aliquot for gene expression assays.

Literature

Brochures