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miTarget™ 3′ UTR miRNA Target Clones

Introduction

GeneCopoeia offers genome-wide human, mouse and rat microRNA (miRNA) 3' UTR target clones in mammalian expression vectors. miRNA 3' UTR target clones can be used for miRNA target identification and functional validation of predicted targets or to study the regulatory effect of miRNA on target gene(s).

 

3' UTR sequences were obtained from public domain gene sequence databases and inserted downstream of the secreted Gaussia luciferase (GLuc) reporter gene in its respective vector system, driven by SV40 promoter for expression in mammalian cells. A chimeric mRNA is transcribed consisting of the GLuc and a 3' UTR target sequence. Thus, the study of mRNA-miRNA target interaction can be easily performed with a live cell assay for GLuc using only 10 μl of the cell culture medium.

Besides using GLuc as the miRNA 3' UTR target reporter, a secreted Alkaline Phosphatase (SEAP) reporter driven by a CMV promoter, is also cloned into the same vector (pEZX-MT05) and serves as the internal control. The dual-reporter vector system enables transfection-normalization for accurate across-sample comparison.

In addition to the GLuc/SEAP dual-reporter vector system, a firefly/Renilla Duo-Luciferase reporter vector (pEZX-MT01) is also available.

mirna target with luciferase reporter

Figure 1. How 3’UTR clone works

 

mirna target with luciferase reporter

Figure 2. Vector backbone of miTarget miRNA 3' UTR target clones

 

 

Figure 3. The inhibitory effect of miRNA on a 3' UTR target sequence expression clone 

LIN28 is the known target gene for miR-125a. HEK 293 cells were transfected with LIN28 3' UTR target sequence expression plasmid alone (left bar) or co-transfected with miR-125a precursor expression plasmid (right bar). Both the GLuc activity and an internal control SEAP activity were determined 24 hours post-transfection. The activity ratio of GLuc to SEAP was set to 1 for the single transfection sample with LIN28 3'-UTR target sequence expression clone (left bar), against which the activity of the co-transfection sample was compared to. The result shows that miR-125a suppressed the luciferase activity from the GLuc-LIN28-3'-UTR clone by more than 70% (right bar).

 

Advantages

 

Live cell assays

  • Naturally secreted GLuc reporter
  • No lysis of the cells is necessary
  • Save samples, reduce variations, and simplify experiments

Dual-reporter vector system

  • Enables transfection-normalization for accurate across-sample comparison

High-throughput compatible

  • Group study or high sample number compatible

High sensitivity

  • GLuc is 1000-fold more sensitive than firefly or Renilla luciferase

Convenience

  • All 3' UTR target clones are transfection-ready

Vectors and Assay Kits

3' UTR target vectors and compatible assay kits                         

Vector Reporter gene Tracking gene Advantage Compatible assay kit
pEZX-MT05 Gaussia luciferase Secreted alkaline phosphatase (SEAP) Live cell assays Secrete Pair ™ Dual Luminiscence Assay Kit
pEZX-MT01 Firefly luciferase Renilla luciferase Cell lysis required Luc-Pair™ Duo-Luciferase Assay Kit

 
Note:
 For miRNA target validation using GeneCopoeia's pEZX-MT05 and pEZX-MT01 target clones, our Secrete-Pair™ Dual Luminescence Assay Kit and Luc-Pair™ Duo-Luciferase Assay Kit are the recommended assay kits respectively. Click here for more information regarding validation of pEZX-MT01 clones.

To Order

Pre-designed 3' UTR target clones

Search over 18,000 human, 18,000 mouse, and 10,000 rat pre-designed 3' UTR target clones in your choice of vector.

 

Custom services

Need a custom 3' UTR target clone? Request a quote here or email inquiry@genecopoeia.com.

 

Download warranty
 MiTarget™ MicroRNA 3' UTR Target Clone Cancellation, QA, License, and Warranty

 

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Publications

 

2012

 

View more miRNA publications