The pmirGLO Dual-Luciferase miRNA Target Expression Vector(a–d) is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites 3´ of the firefly luciferase gene (luc2 ). These target sites can be introduced by cloning putative miRNA binding sites alone, or the 3´ untranslated region (UTR) of a gene of interest, to study the influence of these sites on transcript stability and activity. Firefly luciferase is the primary reporter gene; reduced firefly luciferase expression indicates the binding of endogenous or introduced miRNAs to the cloned miRNA target sequence. This vector is based on Promega dual-luciferase technology, with firefly luciferase (luc2 ) used as the primary reporter to monitor mRNA regulation and Renilla luciferase (hRluc-neo) acting as a control reporter for normalization and selection. This vector contains the following features:

• Human phosphoglycerate kinase (PGK) promoter provides low translational expression, which is advantageous when reduction of signal is the desired response. The PGK promoter is a nonviral universal promoter, which functions across cell lines (yeast, rat, mouse and human).
• Firefly luciferase reporter gene (luc2 ) inversely reports miRNA activity in mammalian cells.
• Multiple cloning site (MCS) is located 3´ of the firefly luciferase reporter gene (luc2 ).
• Humanized Renilla luciferase-neomycin resistance cassette (hRluc-neo) is used as a control reporter for normalization of gene expression and stable cell line selection.
• Ampr gene allows bacterial selection for vector amplification.
• SV40 late poly(A) signal sequence is positioned downstream of luc2 to provide efficient transcription termination and mRNA polyadenylation.
• Synthetic poly(A) signal/transcription stop site.