◎ Excellent cell transfection performance: the positive rate of cell transfection is over 90%, the gene knockout effect is obvious, and the knockout efficiency of laminin A/C gene in A549 cells is over 95%;
Very low cytotoxicity: the death rate of transfected cells is less than 10%, which greatly reduces the influence of cytotoxicity on experimental results;
◎ The transfection range is wide, and most adherent cell lines can obtain ideal transfection results.
Product introduction
RFect small nucleic acid transfection reagent is a new type of small nucleic acid transfection reagent successfully developed by our research and development team led by internationally renowned scientist Dr. Cui Kunyuan in Seattle Laboratory, USA. RFect can be used to transfect small molecular RNA and DNA within 200bp, such as siRNA, antisense RNA, microRNA, etc. Transfected cells include most adherent cells, such as common cell lines and tumor cell lines. At present, the main components of transfection reagents are liposomes or polyethyleneimine (PEI), both of which have great cytotoxicity and poor transfection effect. RFect adopts a new type of animal-derived nanomaterial, which has low toxicity and excellent transfection performance. Compared with other brands of small nucleic acid transfection reagents, the cell transfection positive rate of RFect is generally above 90%, and the inhibition efficiency of laminin A/C gene is above 95%, while the cell transfection positive rate of other brands of transfection reagents is generally less than 70%, and the inhibition efficiency of laminin A/C gene is less than 75%. The cytotoxicity of RFect is very low, and the death rate of transfected cells is less than 10%, while the death rate of transfected cells of other brand reagents is generally above 30%. Serum has no effect on transfection effect, so it is not necessary to add or change the culture medium deliberately. We have applied for an international patent on the material synthesis and reagent preparation of RFect small nucleic acid transfection reagent, and covered many countries and regions in the world through PCT. This product is suitable for cell line transfection and primary cell transfection. Please select RFectPM small nucleic acid transfection reagent.
Operating Steps: This instruction is suitable for transfection experiments of 24-well culture plates. For the dosage of culture plates of other specifications, please refer to the following table, which shows the dosage and volume of each hole.
A. Cell inoculation: Inoculate cells one day before transfection, with 500 μl culture medium per well, so that the cell density is 30-50% during transfection, and try not to use antibiotics.
B. preparation of B.SiRNA-rfect mixture:
1.6pmol siRNA was diluted with 50μl serum-free medium.
2.2μl reagent was diluted with 50μl serum-free medium. Gently for 5 minutes and incubate at room temperature for 5 minutes. Note: Be sure to complete the third step within 25 minutes, and don't delay too long.
3. After incubating for 5 minutes, incubate siRNA diluent with RFect diluent (total volume 100μl) for 5 minutes. Gently mix and incubate at room temperature for 20 minutes.
C. adding the mixture to the cultured cells (complete medium culture):
1. Add the mixture of 100μl into a culture well containing 0.5ml of cultured cells. Shake the Petri dish gently and mix well.
Culture at 2.37℃ 18-72h, and detect the gene inhibition effect. If necessary, the medium can be changed after 4-6 hours of cell culture, but it is not necessary. The incubation time depends on the cell type.
Interference gene itself and analysis method. Different incubation times can be set for the experiment to determine the best incubation time.
Optimization of transfection experiment: In order to improve transfection efficiency, it is suggested to optimize transfection conditions, especially the first transfection. For example: 24-well Petri dish, adjustment? The dosage of siRNA and reagent. The dosage of siRNA is adjusted between 0.6-30 pmol (final concentration 1-50 nm), and the dosage of RFect reagent is adjusted between1.0-3.0 μ l. Customers can conduct pre-experiments according to the conventional dosage. If the experimental results are not satisfactory, the dosage of transfection agent can be adjusted and explored appropriately.
Key points of transfection experiment:
L Try not to use antibiotics during transfection, otherwise it will lead to increased cell death;
L The dose of siRNA in the first experiment was set to the final concentration of 10nM, 30nM, 50nM, 100 nM, and the subsequent experiments were revised according to the experimental results.