Human TLR3 TranslationBlocker™ siRNA Duplex Product Attributes
Blocks transcription and translation of TLR3 from: Human.
May also react with TLR3 based upon sequence homology and clone specific publications.
Type of RNAi: siRNA.
siRNA target sequence: NM_003265.
Samples and cell lines tested: A549 Human Adenocarcinoma cells from Basal Alveolar Epithelium.
Buffer and Stabilizer: Lyophilized. See datasheet for resuspension instructions and buffer information.
Concentration of Anti-Human TLR3: 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.
TLR3 Knockdown Protocol:
- Starting with cultured cells in growth media seed plates with cells and incubated at 37°C. The above data was generated in 24 well plates with 5x104 cells incubated at 37°C until cells reached 70% 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 2000 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 24 well plates, we recommend starting siRNA dose optimization at around 50 pmol siRNA duplex.Optimal dilution should be experimentally determined.
- Combine diluted siRNA and transfection reagent. Ratio of transfection reagent to siRNA should be optimized. Equivalent volumes of prepared Lipofectamine 2000 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 24 well plates this will entail adding the solution to cells in approximately 250 ul 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. Generally, a 4 hour incubation or initial transfection incubation is recommended.
- 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. TLR4 knockdown at the protein level was measured at 48 hours post transfection via ELISA. Infection of cells with RSV was performed following transfection to stimulate TLR3 expression
TLR3 General Information
Gene Name: TLR3
Entrez Gene ID: 7098
NCBI Ref Seq Accession Number: NP_003256.1
TLR3 Molecular Weight:
Alternate Names: TLR-3, Toll-like Receptor 3, CD283
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.