There continues to be some unpredictability in the use of RNA
interference (RNAi) for gene silencing, which often requires
time-consuming effort to determine effectiveness, select the best
constructs, and better optimize use in knockdown applications. Testing
requires the use of antibodies (which may not be available), expensive
Q-PCR probes, phenotypic measurements, or expensive equipment.
To solve this problem OriGene has created a fusion construct, which incorporates a full-length OriGene TrueClone and a luciferase reporter gene, the RNAi Validation Vector.
Using this new tool it is possible to measure the effectiveness of RNAi constructs with targeted specificity using nothing more than a luminescence reader. With the RNAi Validation Vector one can quickly find the effective knockdown construct as well as the optimal transfection conditions.
Compatible with any RNAi technology Currently there are three RNAi gene-silencing platforms that can trigger an RNAi response, short interfering RNA (siRNAs), short hairpin RNA (shRNA), and micro RNA (miRNA) constructs. The RNAi Validation Vector can measure the effectiveness of knockdown using any of these RNAi platforms.
Using different restriction sites, the gene of interest is cloned into the vector 3’ to the firefly luciferase gene. The construct is then transfected into the mammalian cell of choice where a chimeric construct is transcribed. Co-transfection of HuSH shRNA, or other RNAi, targeting the gene of interest produces siRNA, which binds to the target mRNA initiating the RNAi process. The firefly luciferase-target gene transcript will be degraded reducing the measurable luciferase, indicating the potency and specificity for RNAi against the target gene.
- Easy to use - Any OriGene TrueClone can be sub-cloned into the RNAi Validation Vector with a simple NotI digest and ligation.
- High throughput application - Because sub-cloning is so simple, this reporter system can be used in optimization for multiple genes and cell lines at a time.
- Reliable detection - The initiation of RNA interference toward the gene of interest in the reporter construct leads to degradation of a fusion mRNA (luciferase / Luc) which results in a measurable decrease in the luciferase signal, indicating a functional RNAi effect.
The firefly luciferase coding region was cloned into OriGene’s pCMV6-XL5 vector using the Xba I cloning site. The 5’ Not I site on the original pCMV6-XL5 vector was mutagenized, leaving a different Not I cloning site downstream of the luciferase cDNA. The full sequence of this vector can be downloaded.