GeneCopoeia offers genome-wide microRNA 3'UTR target constructs in mammalian expression vector systems with luciferase reporter genes for functional validation experiments.
23,000 human
19,000 mouse
8,000 rat
Choose the right luciferase vector depending on your assay requirements
1.
Perform assay on cell lysates
— with Firefly luciferase
2.
Perform assay without destroying
target cells — with Gaussia luciferase
Vector
Reporter gene
Tracking gene
Advantage
pEZX-MT01
Firefly luciferase
Renilla luciferase
Perform assays on cell lysates
pEZX-MT02
Gaussia luciferase
Alkaline phosphatase
Perform enzymatic assays without destroying target cells
Features of microRNA 3’ UTR target clone collections
All 3’ UTR sequences (inserted downstream of coding sequences) were obtained from public domain gene sequence databases.
Expression cassettes are inserted downstream of the firefly or Gaussia luciferase reporter genes which are controlled by the SV40 promoter for expression in mammalian cells.
The Renilla luciferase or alkaline phosphatase genes can be used as a tracking indicator for successful transfection and expression of the miRNA constructs in target cells.
The regulatory effect of a particular miRNA on its potential target is assessed with a functional assay for the firefly or Gaussia
luciferase.
Expression plasmids transcribe a chimeric mRNA consisting of the firefly or Gaussia luciferase coding sequence and a 3’ UTR target sequence.
Luciferase expression is regulated by binding of the targeting miRNA to the 3’ UTR target sequence and luciferase activity is quantified with a colorimetric assay.
Applications of precursor miRNA and target expression clones
Gene expression regulation — The mRNA regulation effect of miRNAs can be studied individually or collectively by transducing precursor miRNAs into target cells with transfection or viral infection.
miRNA target gene identification and validation — Together with the miTarget miRNA target validation expression clones, miRNA expression constructs can be used to identify and verify their target genes.
Luc-Pair™ miR Luciferase Assay Kit - optimized luciferase reporter assay for use with GeneCopoeia miRNA Target Sequence 3' UTR Expression Clones
The Luc-Pair™ miR Luciferase Assay Kit provides an efficient system in a convenient 96-well plate format for measuring firefly and Renilla luciferases sequentially. Optimized for use with GeneCopoeia miRNA 3' UTR Target Sequence Expression Clones, Luc-Pair™ miR assays enable easy, cost effective validation of miRNA activity.
Luc-Pair™ miR Luciferase Assay provides the following advantages:
Figure 3. The inhibitory effect of miRNA on a target sequence (3’ UTR) expression clone can be measured with GeneCopoeia Luc-Pair™ miR Luciferase Assay kit.HEK 293 cells were plated on a 6-well plate. On the second day, the cells were transfected with 1.0 µg of Lin28 3’ UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc (Product ID: HmiT019205) and 1.4 µg of miR-125a precursor miRNA expression clone in pEZX-MR03 vector with eGFP (Product ID: HmiR0309) and miRNA control clone as indicated in the figure. The cells were transferred to a 96-well plate 18 hours after transfection and cultured for another 24 hours. Both firefly luciferase and Renilla luciferase activities were measured and data was recorded on Victor II machine. Firefly luciferase activity was then normalized with Renilla luciferase activities in the same well.
Figure 4. Data showing the effect of miArrest miRNA inhibitor clone in a dose-dependent manner.A miR-125a inhibitor expression plasmid (GeneCopoeia HmiR-AN0094-AM01) was transfected into HEK 293 cells with 1) a miR-125a precursor expression plasmid (GeneCopoeia HmiR0309-MR03) and 2) a miRNA target sequence expression clone expressing LIN28, a known target gene for miR-125a (GeneCopoeia HmiT019205-MT02: 3’-UTR sequence of LIN28 in gaussia luciferase-alkaline phosphatase dual reporter expression vector). Both the GLuc activity and an internal control AP activity were determined 24 hours post-transfection. The activity ratio of GLuc to AP was set to 1 for the single transfection sample with LIN28 3’-UTR target sequence expression clone (left-most bar), against which the activities of other samples were normalized. The result shows that mir-125a suppressed the luciferase activity from the Gluc-LIN28-3’-UTR clone by more than 70% (Bar 2 from left). This suppression effect was blocked by the introduction of varying amount of miArrest™ inhibitor clone against miR-125a in a dose-dependent manner. At the highest dose, the reporter GLuc activity is higher than the control (right-most bar). This could be attributed to the fact that this vector-based inhibitor may have blocked the regulatory effect of endogenous miR-125a, which would result in increased translational activity of GLuc-LIN28-3’-UTR transcript.
