1. How are 27-mer Dicer-Substrate siRNA different from traditional 21mer siRNAs?
Traditional 21-mer RNA duplexes mimic the natural siRNAs that are the result of Dicer processing of long substrate RNAs. In contrast, 27-mer siRNAs go through the natural mechanism of Dicer processing to produce 21-mer siRNAs. The Dicer-substrate approach can result in 10-fold higher potency than traditional 21-mer siRNAs targeted to the same site
2. What are the benefits of the Trilencer-27 siRNA duplex design?
A full screening was performed which included blasting the sequences to ensure they only align to the target gene, performing secondary structure checks, and confirming duplex stability for incorporation into the RISC complex
3. How should I use the products?
Customers can use the 3 siRNA duplexes directly for transfection and gene-knockdown studies. After transfection, cell lysates can be obtained and used for Western blot analysis with an antibody against the target protein to verify the functionality of the siRNA duplex, or RNA can be harvested from transfected cells and used in quantitative RT-PCR to determine the loss of gene expression. If desired, the validated siRNA target can be integrated into OriGene’s shRNA viral vectors using exact-shRNA service . OriGene shRNA viral vectors include retroviral, lentiviral and AAV vectors that can be used for transfection and/or viral infection. The shRNA plasmids can be re-transformed for unlimited supply.
4. What controls are offered with the Trilencer-27 kit?
The control we offer with the kit is Universal scrambled negative control duplex (SR30004).
Other controls are available for purchase and are listed below:
- TYE 563-labeled transfection control RNA duplex (SR30002)
- HPRT1 Positive Control duplex (SR30003)
5. What use does the scrambled non-effective siRNA duplex serve?
To specifically rule out the potential non-specific effect induced by expression of the siRNA, OriGene provides customers with a negative control (SR30004). The duplex should serve as a negative control for gene-specific knockdown experiments and exclude any potential interferon response.
6. Does the siRNA kit come with RT-PCR reagents?
The siRNA kit includes:
- 3 Dicer-Substrate RNAi duplexes, 2 nmol each
- 1 negative control duplex, 2 nmol
- Rnase-free duplex re-suspension buffer
7. What is the concentration of Trilencer-27 siRNA needed to conduct the experiment and see knockdown?
The actual level of target gene knockdown relates to the transfection efficiency. A positive control such as HPRT siRNA should always be used in each experiment to assess transfection efficiency. In addition, varying amounts of Trilencer-27 duplexes ranging from 0.1 to 50 nM can be used to determine which siRNA amount shows maximum knockdown. Normal optimization concentrations vary between 1-50nM siRNA, with 10nM being most common
8. What is OriGene’s guarantee on siRNA duplexes?
OriGene guarantees that at least two of the three Dicer-Substrate duplexes in the kit will provide at least 70% or greater knockdown of the target mRNA when used at 10 nM concentration by quantitative RT-PCR when the TYE-563 fluorescent transfection control duplex (cat# SR30002) indicates that >90% of the cells have been transfected and the HPRT positive control (cat# SR30003) provides 90% knockdown efficiency.
For non-conforming siRNA, requests for replacement product must be made within ninety (90) days from the date of delivery of the siRNA kit. To arrange for a free replacement with newly designed duplexes, please contact Technical Services at firstname.lastname@example.org. Please provide your data indicating the transfection efficiency and measurement of gene expression knockdown compared to the scrambled siRNA control (quantitative RT-PCR data required).
9. Trilencer-27 siRNAs require a special transfection reagent?
Yes, it is important that a transfection reagent designed for siRNA transfection be used. OriGene recommends siTran 2.0 (TT320001) because it can be used for siRNA alone or co-transfection of siRNA and plasmid expression constructs
10. How should I cite your product?
Here is our guideline:
Full Product Name, OriGene Technologies Inc., Rockville, MD, USA, Catalog #, Lot #.
In addition, we would love to hear from you when the paper is published. Email us a copy of the accepted manuscript and receive a special gift.