RNAi Validation Vector FAQs

Can I use the validation system with other RNAi products like siRNA oligos?
Answer: The pCMV-LUC(V) system is appropriate for screening siRNA, shRNA, or miRNA constructs. You must be certain to subclone into the validation vector the region of the cDNA targeted by your RNAi constructs; for example, if your siRNA targets the 5’ UTR, then you must clone into pCMV-LUC(V) the cDNA including 5’ UTR.

Does this system produce a luciferase – gene fusion protein?
Answer: No. Since the luciferase cDNA cloned into this vector contains its own stop codon, there will be no chimeric fusion protein of luciferase and your target gene produced. However, an intact luciferase protein will be translated if the chimeric transcript is not degraded due to RNAi against the target gene.

Is the coexpressed gene really similar to the gene I’m trying to silence?
Answer: In principal, the chimeric mRNA containing luciferase and your targeting gene should be similar to the endogenous version of your target mRNA. However, since the amounts of overexpressed mRNA may be much higher than endogenous mRNA, it may be more difficult to knockdown your target gene as part of the chimeric mRNA.

Has the validation system been shown by other means that it provides specific quantifiable knockdown?
Answer: OriGene is currently testing this and we would also appreciate if you could provide us with your own experimental results of this comparison.

Can I subclone genes that are not from the OriGene TrueClone collection into the validation vector?
Answer: Yes, you can subclone any cDNA into this vector in order to determine the potential knockdown effect of shRNA/siRNA constructs.

Other than Not I, what other MCS restriction sites are available for subcloning?
Answer: Sac II and Sma I are also available at the MCS. Please confirm that these restriction sites do not exist in the internal region of the cDNA before attempting to clone into this vector.

Which cloning methods work best with the pCMV-LUC(V) vector?
Answer: Non-directional Not I subcloning and restriction and PCR-based directional subcloning are equally compatible with this vector. It is the customer’s preference which to choose. Of course, Not I, Sac II, and/or Sma I are available for directional cloning, this is the simplest strategy.

How can I subclone a Gateway compatible clone into this vector?
Answer: Determine if there are any cloning sites in the Gateway vector compatible with pCMV-LUC(V). If not, use the PCR directional cloning strategy (see protocol section) to clone the Gateway cDNA into pCMV-LUC(V).

Do I have to cotransfect the shRNA and the validation vector or should they be introduced to the cells separately? Which would be best?
Answer: We recommend that you cotransfect both pCMV-LUC(V) and shRNA plasmids simultaneously. Commercially available transfection reagents, such as OriGene’s Turbofectin 8.0, work very well for the cotransfection of cDNA and shRNA plasmids.

How does a gene specific shRNA or siRNA end up degrading the luciferase reporter in this system?
Answer: The shRNA/siRNA forms complex with DICER in the RISC to degrade the corresponding mRNA sequence to be targeted. This RNAi effect can be extended 5’ to the targeting sequence into the luciferase mRNA, leading to the downregulation of luciferase protein activity.

What advantages does this system have over other similar reporter systems?
Answer: Our system is particularly suitable for the subcloning of OriGene cDNA clones into this vector system as the Not I sites exist on both vectors. Moreover, our vector system utilizes firefly luciferase for the measurement, whose substrate is readily and commercially available, unlike other forms of luciferase.

Does this replace the use of antibodies for Western blot analysis or qRT-PCR knockdown detection?
Answer: No. This system is recommended as a prescreening method to choose the most effective construct with which to do your experiments. It is still advised to perform Western blot analysis, qPCR, or functional assays to monitor the level of gene knockdown.

Once I have determined which knockdown construct works best, should I keep or discard the others?
Answer: One construct may work better in one cell line and another may work best in a different cell line. If you change cells types it may be necessary to rescreen to find the best performer. The other constructs may also be useful for partial knockdown studies.

What luciferase substrate would you recommend for use with the RNAi Validation System?
Answer: We typically use Perkin Elmer’s Ultra-High Sensitivity Luminescence Reporter Gene Assay System (No. 6016977).

What advantages does this system offer in the design of large screening assays?
Answer: If OriGene TrueClones are used in the design, one can subclone many genes into this vector at once using Not I. One can even mix the purified inserts (different cDNAs) before ligating them into the vector. In this manner, one can clone many cDNAs into this vector in a very quick period of time. Bulk clone pricing is available; contact OriGene’s Customer Service (custsupport@origene.com, 888-267-4436 or 301-340-3188) for more information.

Can the validation vector itself interfere with gene expression in my cells?
Answer: We do not have data to exclude this possibility. Thus, it is always very critical to include suitable control DNAs in the cotransfection experiments.

If my validation vector experiments show incomplete knockdown, does this mean that my RNAi constructs aren’t effective?
Answer: No, not necessarily. This validation system is capable of identifying the most effective construct from a set, but should not be used to exclude any one particular construct. This system is best used to determine the most effective construct as a pre-screening tool. However, we don’t have a sufficient amount of data as yet to indicate that our validation vector system fully correlates with the outcome of using other methods for shRNA/siRNA validation at this time.

Does OriGene provide any guarantee of knockdown in subsequent experiments when an RNAi construct has been validated using this method?
Answer: No. The degree of knockdown of your target gene will be dependent on a number of factors, including transfection or infection method and efficiency, target cells, and transcript or protein half-life. For this reason, OriGene cannot guarantee knockdown using a pCMV-LUC(V) validated construct.