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Comprehensive solutions for microRNA studies

Find your miRNA Expressway to Discovery at GeneCopoeia with the largest collection of miRNA clones, target validation constructs, luciferase reporter assays, validated qPCR miRNA primers and qRT-PCR miRNA detection kits.

GeneCopoeia microRNA comprehensive analysis tools enable efficient and fast protocols for investigating miRNA functions in cell culture.

*Not sure which combination of products is right for your project? Contact us with a few details and we will provide recommendations especially designed for you.

miExpress™ Precursor miRNA Expression Clones

GeneCopoeia offers precursor miRNA expression constructs in a feline immunodeficiency virus- (FIV) based lentiviral vector system. An RNA polymerase III type promoter (H1) is used to drive transcription of hairpin precursor miRNAs (approximately 150 nucleotides), which are then processed to generate mature miRNAs by RNAi enzymes. The EGFP tracking reporter gene is co-expressed with the neomycin selection gene under the control of the CMV promoter and bicistronic internal ribosome entry site (IRES) element, providing a convenient method for monitoring miRNA expression, transfection and transduction efficiencies.

GeneCopoeia miRNA constructs are made with proprietary, state-of-the-art technology, are fully sequenced and come with a guarantee ensuring the integrity of critical regions. For you, that means greater confidence and accurate results faster.

RISC-miRNA complexes specifically regulate gene expression by binding to the 3’ UTR of mRNAs of target genes (Fig. 1): 1) translation suppression and 2) mRNA cleavage, degradation, and translation inhibition.

Figure 1. Lentiviral vector based precursor miRNA expression clones and the mechanism of vector mediated miRNA gene regulation

Features of Precursor miRNA Expression Clone Collections

• All known human, mouse and rat miRNA in release 13.0 in miRBase are available
• Fully-sequenced expression cassettes of all miRNA expression constructs
• Lentiviral-based expression constructs allow efficient infection-based transduction of miRNAs into non-dividing and otherwise difficult to transfect target cells as well as conventional transfection-based transduction into dividing cells.
• A stable cell-line selection marker (neomycin) enables regulation studies of both long-term and transient expression
• Efficiencies of vector-plasmid transfection and virus-infection transduction are monitored with the EGFP reporter protein.

miTarget™ miRNA Target Sequence 3' UTR Expression Clones

GeneCopoeia offers genome-wide miRNA target sequence (3’ UTR) expression constructs in a mammalian expression vector system. All 3’ UTR sequences used in these expression constructs were obtained from public domain gene sequence databases. The 3’ UTR sequences are inserted downstream of coding sequences. These cassettes are inserted downstream of the firefly luciferase reporter gene which is controlled by the SV40 promoter for expression in mammalian cells. Vectors also include the renilla luciferase gene, which can be used as a tracking indicator for successful transfection and expression of the miRNA constructs in target cells. Figure 2 shows the basic features of the expression construct.

The regulatory effect of a particular miRNA on its potential target is assessed with a functional assay for the firefly luciferase. These expression constructs transcribe a chimeric mRNA consisting of the firefly 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. Luciferase activity is quantified with a colorimetric assay.

Figure 2. Vector features for human miRNA target sequence expression clones

Applications of Precursor miRNA and Target Expression Clones

• Gene expression regulation. The mRNA regulation effect of the 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 andRenilla 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 mg of target sequence (3’ UTR) expression clone (pEZX-MT01-Lin28 UTR-fLuc) and 1.4 mg of miRNA expression vector (or miRNA control vector) 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.

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