The transfection performance of suspension cells is excellent: the positive rate of cell transfection is generally above 75%, and the gene knockout efficiency is above 80%;
Very low cytotoxicity: the death rate of transfected cells is less than 10%, which greatly reduces the influence of cytotoxicity on experimental results;
◎ Transfected cells have a wide range, and most suspension cells can obtain ideal transfection results.
Product introduction
RFectSP small nucleic acid transfection reagent for suspension cells is a special reagent for small nucleic acid transfection of suspension cells, which was successfully developed by our research and development team on the basis of RFectPM primary cell small nucleic acid transfection reagent. RFectSP can be used to transfect small molecular RNA and DNA within 200bp, such as siRNA, antisense RNA and microRNA, and can transfect most suspension cells. At present, nucleic acid transfection in suspension cells is a hot issue at home and abroad, and there is still a lack of truly effective commercial transfection reagents for small nucleic acids in suspension cells in the market. RFectSP can efficiently transfect most suspension cells and obtain ideal gene knockout effect. For most suspension cells, the transfection positive rate of RFectSP is over 75%, while the death rate of transfected cells is less than 10%. The use of RFectSP is also very simple. Firstly, sRNA and RFECTSP were mixed at room temperature, and then the mixture of SiRNA and RFectSP was directly added to the cells containing culture medium. Serum has no effect on transfection effect, so it is not necessary to add or change the culture medium deliberately. We have applied for the international patent of material synthesis and reagent preparation of RFectSP suspension cell small nucleic acid transfection reagent, and covered many countries and regions in the world through PCT.
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-rfectsp mixture:
1.6pmol siRNA was diluted with 50μl serum-free medium.
2.2μl RFectSP 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 incubation for 5 minutes, the SiRNA was diluted with RFectMN 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. Gently shake the culture plate for 5 minutes and fully shake it for 5 minutes.
Cultured at 2.37℃ for 48-72h, and the gene inhibition effect was detected. 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 best to optimize transfection conditions, especially for the first time. For example: 24-well culture plate, adjust the dosage of siRNA and RFectSP reagent. The dosage of siRNA was adjusted between 6 and 6-60 pmol (final concentration 10- 100 nm), and the dosage of RFectSP reagent was adjusted between1.0-3.0 μ l.
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.