Short interfering RNA (siRNA) can be directly introduced into cells by transfection. A lipid-based transfection reagent, such as X-tremeGENE siRNA Transfection reagent, can provide a convenient, reliable, and efficient vehicle for delivering siRNAs into animal cells, enabling the study of cellular and functional consequences of gene knockdown.
This innovative reagent forms a complex with siRNA, as well as with mixtures of siRNA and plasmid DNA (cotransfection), and efficiently delivers the nucleic acids into animal cells to induce gene silencing. Transfection is achieved in just a few steps: mix and incubate diluted transfection reagent with diluted siRNA, then add this complex to cells. Because X-tremeGENE siRNA Transfection Reagent functions exceptionally well in the presence or absence of serum and demonstrates low cytotoxicity, it can be used without media changes. The product is animal-component free.
Solution filtered through 0.2 μm pore size membrane, supplied in polypropylene tubes.
Activity assay: X-tremeGENE siRNA Transfection Reagent (1 - 2.5 μl) is combined with siRNA (0.1 - 0.35 μg) that is specific for the HPRT housekeeping gene. The mixture is used to transfect HEK-293 cells (in a monolayer, 30 - 50% confluent) in the presence of 10% fetal bovine serum (FBS). Following transfection, the decrease of HPRT mRNA cells is determined with the LightCycler® Real-Time PCR System. A knockdown efficiency of 70 - 95% is typically observed when mRNA is measured.
Cytotoxicity analysis: HEK-293 cells are exposed to siRNA/X-tremeGENE siRNA Reagent complexes for 72 hours in the presence of serum, without a media change. Cytotoxicity is then tested by analyzing the cells with the WST-1 Cell Proliferation Reagent (Roche).