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In Vitro siRNA Transfection Reagents:
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, has a well-defined structure: a short (usually 21-nt) double strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. siRNAs can be introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important and indispensable tool for gene function and drug target validation studies in the post-genomic era.
While liposome or polymer reagents often give very good DNA delivery efficacy, they often failed to efficiently drive siRNA into mammalian cells due to the length of the siRNA anionic segment that is too short to maintain electrostatic cohesion with the cationic lipids or polycationic polymer. We modified the liposome and polymer by addition of specific pre-screened hydrophobic groups which confers the pH dependent conformational changes (PDCC) at physiological pH condition and greatly stabilizes siRNA lipolplex or polyplex.
Based on our unique and proprietary “PDCC” technology, currently we are developing and manufacturing the following four siRNA transfection reagents, which show distinct transfection characteristics with the leading products in the market.
siRNA Transfection Reagent Selection Chart.
PepMute™ Plus siRNA & DNA Transfection Reagent is an upgraded version of PepMute™ reagent (Cat # SL100566) by crosslinking pre-screened hydrophobic amino acids to its backbone. With our proprietary pH Dependent Conformational Change (PDCC) technology, the transfection reagent which contains 36 amino acids long's peptide backbone was formulated by addition of pre-screened hydrophobic amino acids to all amine groups, conferring PepMute™ Plus reagent an ability to self-assembly during formation of transfection complex and making PepMute™ reagent a versatile and most powerful gene delivery tool which provides more than 95% silencing efficiency at 1 nM siRNA in variety of mammalian cells. PepMute™ Plus Reagent have been validated to effectively and reproducibly transfect single DNA or siRNA or co-transfect DNA/siRNA to variety of mammalian cells.
Figure 1. A cartoon showing PepMute™ Plus siRNA Transfection Reagent was developed
Size & content:
- PepMute™ Plus Transfection Reagent, 1.0 ml, sufficient for ~1000 reactions based on transfecting 2.5 pmols siRNA in 24-well plate
- PepMute™ Plus Transfection Buffer(5x ), formulated for maximal transfection efficiency, 8.0 ml
Store at 4 °C and never freeze. If stored properly, the product is stable for 12 months or longer.
- Effective knockdown (95%) with only 0.5 pmol siRNA (1.0 nM siRNA in 24-well plate)
- Formulated for hard-to-transfect mammalian cells
- One tube reaction and easy transfection protocol
- Best for broad spectrum especially hard-to-transfect mammalian cells
- Very low cytotoxicity
Comparisons of Knockdown Efficacy of PepMute™ Plus siRNA & DNA Transfection Reagent with Brand Name Products
Figure 2. Knockdown efficacy comparison of PepMute™ Plus Transfection Reagent (upper panel) vs. Dharmafect™ 4 siRNA Transfection Reagent (middle panel) and Lipofectamine™ 2000 (lower panel) on HEK293 cells. siRNA targeting renilla luciferase at different final concentrations ranging from 0.5 to 20 nM was co-transfected with renilla luciferase gene (0.5 µg of pRL-CMV DNA per well) by the above three transfection reagents per manufacturers' protocols into HEK293 cells growing on a 24-well plate. Renilla luciferase activity was determined 24h after post co-transfection with renilla luciferase determination system (Promega). The luminescence was measured from 5.0 µl of lysate during 10s integration with a luminometer (Beckman Coulter LD 400). Luciferase activity was expressed as light units integrated over 10s (RLU) and normalized per mg of cell protein by using the BCA assay. The errors bars represent standard deviation derived from triplicate experiments. Luciferase-silencing efficiency was calculated relative to untreated cells. While PepMute™ Plus and Dharmafect™ 4 reagents delivered significant gene silencing from 1.0 nM of renilla luciferase siRNA, lipofectamine™ 2000 gave good knockdown only after 30 nM (data not shown).
Figure 3. PepMute™ Plus Transfection Reagent knocked down GFP gene expression in HepG2 cells. GFP cDNA, pEGFP-N3, was co-transfect with a siRNA targeting GFP gene (final 5.0 nM, right panel) and a sham siRNA (final 5.0 nM, left panel) into HepG2 cells by PepMute™ Plus Transfection Reagent. GFP fluorescence was visualized 24h post transfection with a Nikon T2000 fluorescence micorscopy. Quantitative analysis showed that GFP siRNA at 5.0 nM delivered by PepMute™ Plus Transfection Reagent knocked down 96% co-transfected GFP expression in HepG2 cells.
Data Sheet & Protocol
- A Protocol for DNA/siRNA Co-Tranafection to Mammalian Cells
- A Protocol for siRNA Transfection to Mammalian Cells
- A Protocol for DNA Transfection to Mammalian Cells
Note: All prices in US Dollars