Human VEGF TranslationBlockerâ¢ siRNA Duplex Product Attributes
Blocks transcription and translation of VEGF from: Human.
May also react with VEGF based upon sequence homology and clone specific publications.
Type of RNAi: siRNA.
siRNA target sequence: NM_003376.
Samples and cell lines tested: Pc-3 Human Prostate Adenocarcinoma Cells.
Buffer and Stabilizer: Lyophilized. See datasheet for resuspension instructions and buffer information.
Concentration of Anti-Human VEGF: Lyophilized
SiRNA Purification Method:RPC/HPLC
Storage Conditions: Store Lyophilized siRNA duplex at 4C. Reconsituted product may be stored at -20C for up to 1 month.
VEGF Knockdown Protocol:
- Starting with cultured cells in growth media seed plates with cells and incubated at 37°C. The above data was generated in 6 well plates with 2x105 cells incubated 20 hours at 37°C until cells reached 70-80% confluence prior to starting transfections
- Prepare transfection reagent in serum-free growth medium such as Opti-MEM-I. Allow to come to room temperature. Lipofectamine with PLUS was used to generate the above data in accordance with manufacturerÂs instructions. li>
- Dilute siRNA to working concentration in serum-free growth media. For Lipofectamine or similar transfections into 6 well plates, we recommend starting siRNA dose optimization at around 100 pmol siRNA duplex.Optimal dilution should be experimentally determined.
- Combine diluted siRNA and transfection reagent. Equivalent volumes of prepared Lipofectamine or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 30 minutes with gentle mixing (optional).
- Add prepared siRNA solution to cells. For 6 well plates this will entail adding the solution to cells in approximately 2.5 ml serum-free medium. Optionally, FITC labeled control siRNA (Product Number QC-1F) or positive control siRNA (Beta-Actin; Product Number QC-2) may be used to assess transfection efficiency.
- Incubate for several hours in serum free media. The above data was generated with a 4 hour incubation.
- Add serum containing media to culture without a media change if longer incubation times are required. Generally 1-2 fold of the original volume of siRNA containing serum-free media is added to restore nutrient levels in media. Alternatively, the media may be directly supplemented with serum such as 10% FBS.
- Incubate and assay several hours after transfection. Optimal knockdown time should also be experimentally determined. Optionally, a second transfection can be performed 24 hours after the first transfection to enhance knockdown. VEGF knockdown at the protein level was measured at 26 hours post transfection via ELISA.
VEGF General Information
Gene Name: VEGFA
Entrez Gene ID: 7422
NCBI Ref Seq Accession Number: NP_003367
VEGF Molecular Weight:
Alternate Names: MVCD1, VPF, VEGFA, VEGF-A
Limitations and Warranty
This product is for Research Use Only. This product is guaranteed to work for a period of two years when stored at -70C or colder, and one year when aliquoted and stored at -20C.