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The psiCHECK-1 and psiCHECK-2 Vectors are designed to provide a quantitative and rapid approach for initial optimization of RNA interference (RNAi). The vectors enable monitoring of changes in expression of a target gene fused to a reporter gene. In both vectors Renilla luciferase is used as the primary reporter gene, and the gene of interest is cloned into a multiple cloning region located downstream of the Renilla translational stop codon. Initiation of the RNAi process by synthetic siRNAs toward a gene of interest results in cleavage and subsequent degradation of the fusion mRNA. Measuring decreases in Renilla activity provides a convenient way of monitoring the RNAi effect. In comparison with other fusion approaches (e.g., GFP or flag-tags), the Renilla luciferase approach offers more convenient and rapid quantitation with higher sensitivity. The psiCHECK-1 Vector is recommended for use in monitoring RNAi effects in live cells. The changes in Renilla luciferase activity are measured with the EnduRen Live Cell Substrate (Cat.# E6481), which allows continuous monitoring of intracellular Renilla luminescence. The psiCHECK-2 Vector contains a second reporter gene, firefly luciferase, and is designed for endpoint lytic assays. Introduction of firefly luciferase in the psiCHECK-2 Vector allows normalization of Renilla luciferase expression, achieving robust and reproducible results.

The pmirGLO Vector is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites downstream or 3' of the firefly luciferase gene (luc2). 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 luc2 used as the primary reporter to monitor mRNA regulation and Renilla luciferase (hRluc-neo) acting as a control reporter for normalization and selection.