| Product Description |
Aladdin's UltraBio™RNAi Transfection Reagent is a highly efficient and easy-to-use reagent for nanomaterial-mediated sRNA delivery. It demonstrates comparable or superior transfection performance to other commercially available sRNA transfection reagents. It is applicable in the delivery of siRNA, miRNA or other forms of small nucleic acids including single-stranded or double-stranded RNA and DNA into eukaryotic cells. It can also be used for gene therapy and transfection of live animals. The nanoparticle technology of the UltraBio™RNAi Transfection Reagent ensures the reliability and stability of both transient and stable transfections of cells.The UltraBio™RNAi Transfection Reagent is extremely easy to use. Mix the serum-free culture medium, sRNA, and UltraBio™RNAi Transfection Reagent. After incubation at room temperature for 20 minutes, the transfection complex is added directly into the cell culture to achieve convenient transfection.The UltraBio™RNAi Transfection Reagent delivers superior transfection efficiency and reproducible results with improved cell viability. It is designed to efficiently transfect both suspension and adherent cells, particularly for adherent cells that are difficult to transfect. Cells can be collected for examination of protein expression 24-48 hours after transfection without changing the cell culture medium. For some target proteins with long half-lives, a significant decrease in protein level may not be detected until 72-96 hours after transfection with siRNA or miRNA.The UltraBio™RNAi Transfection Reagent has been tested on a variety of cell lines with a transfection efficiency of 80-90%. Those tested cell lines include mouse embryonic fibroblast cells NIH3T3, human embryonic kidney cells HEK293, cervical cancer cells Hela, and ovary CHO cells of the Chinese hamster. For more comparison and selection of transfection reagents for adherent cells, please refer to the following webpage
Precautions： Transfection efficiency may decrease with the increase of LipoRNAi™ Transfection Reagent. Start with the recommended dosage in the protocol and optimize the dosage only when necessary.Use high-quality sRNA to achieve a higher transfection efficiency.Cells must be in an optimum physiological condition for transfection.Changing the culture medium 4-6 hours after transfection does not interfere with the transfection efficiency LipoRNAi™ Transfection Reagent. If necessary, the culture medium can be changed 4-6 hours after transfection.Antibiotic-free and serum-free culture medium, such as Opti-MEM® culture medium or DMEM culture medium, is required but not supplied in this product. Mix the LipoRNAi™ Transfection Reagent gently. Vortex or centrifuge should be avoided.Close the LipoRNAi™ Transfection Reagent tube tightly to avoid the exposure of Transfection Reagent to air, which may reduce the transfection efficiency.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. siRNA transfection:The protocol is optimized for transfection in a 6-well plate format. To transfect cells in other formats of cell culture vessels, adjust the reagent volume proportionally as indicated in the table below.a. The day before transfection (18-24 hours), seed about 20-700,000 cells per well in 6-well plates, depending on the cell type, size and the growth rate of cells. At the time of transfection, the optimal confluency for adherent cells is 70-80%. Suspension cells should be in the logarithmic growth phase at the time of transfection. b. Prior to the following transfection step, replace with 2ml of fresh culture medium (complete medium containing serum and antibiotics) per well. Antibiotics do not affect the transfection efficiency of LipoRNAi™ Transfection Reagent or cause cytotoxicity after transfection.c. For each well of a 6-well plate, mix 100pmol siRNA gently by pipetting with 125µl of antibiotic-free and serum-free DMEM culture medium (either high or low sugar DMEM) or Opti-MEM® Medium in a sterile Eppendorf tube, followed by adding 4µl of LipoRNAi™ Transfection Reagent. Mix well gently by pipetting and incubate at room temperature for at least 20 minutes. Vortex or centrifuge should be avoided. The LipoRNAi™ Transfection Reagent/siRNA complexes are stable for up to 6 hours at room temperature.96-well48-well24-well12-well6-well6cm dish10cm dishSerum-free culture medium or Opti-MEM® Medium5μl12.5μl2.5μl50μl125μl250μl750μlsiRNA4pmol10pmol20pmol40pmol100pmol200pmol600pmolLipoRNAi™ Transfection Reagent0.16μl0.4μl0.8μl1.6μl4μl8μl24μlMix gently after adding siRNA in serum-free medium, add LipoRNAi™ Transfection Reagent, mix gently and incubate for at least 20 minutes at room temperature (Figure 3).Amount of complexes added per well5μl12.5μl2.5μl50μl125μl250μl750μlNote 1: The dose of LipoRNAi™ Transfection Reagent can be appropriately adjusted in the range of 2-6μl for each well of a 6-well plate. siRNA dosage can be adjusted in the range of 50-250pmol. A siRNA (pmol): LipoRNAi™ (μl) ratio of 25:1 is generally used, but can be ranging from 10:1 to 40:1 for optimal transfection efficiency. The ratio in the above table is 25:1 with a lower dosage of LipoRNAi™, which is both economical and efficient. The optimal transfection condition varies depending on the cell type and culture conditions and can be optimized within the range recommended above.Note 2: The recommended concentration of siRNA is 20µM. A siRNA concentration ranging from 10µM to 50µM is generally used.Note 3: Prepare a master mix when transfecting multiple cell samples with the same sRNA at the same amount, and then dispense the recommended amount to each well.Note 4: For other culture vessels, the dose of each reagent can be scaled up or down to the culture area of the cell culture. For the transfection of oligonucleotides or RNA, please refer to the protocol for DNA transfection.Figure 3: The effect of incubation time at room temperature after mixing siRNA with LipoRNAi™ Transfection Reagent on the down-regulation of STIM1 expression. LipoRNAi™ Transfection Reagent/STIM1-siRNA complexes were incubated at room temperature for different periods as indicated in the figure prior to the transfection of Hela cells. Cell samples were harvested 48h after transfection for examination of STIM1 protein level via Western Blot. As shown in the figure, STM1 protein expression is down-regulated significantly in cell samples transfected with LipoRNAi™ Transfection Reagent/STIM1-siRNA mixture pre-incubated at room temperature for 20 minutes prior to transfection.d. Add 125μl of LipoRNAi™ Transfection Reagent/siRNA complexes to each well. Gently rock the plate to ensure an even distribution of the transfection mixture in the well, and continue culture directly without changing the culture medium.e. Incubate at 37℃ in a CO2 incubator for more than 48 hours. Analyze the expression of the target gene by appropriate methods such as qPCR, Western Blot, ELISA, reporter gene, etc.FAQ:1. Low transfection efficiency:a. Optimize the ratio of sRNA to LipoRNAi™ Transfection Reagent. Increase the amount of LipoRNAi™ Transfection Reagent for cells that are difficult to transfect.b. Use highly purified, sterile, and contaminant-free sRNA for transfection. c. Make sure that adherent cells are 70-80 % confluent and suspension cells are in the logarithmic growth phase at the time of transfection. Too high or too low of cell density may result in poor transfection efficiency. The optimal cell density for transfection varies for different cell types and should be determined for every new cell line to be transfected. d. Antibiotic-free and serum-free culture medium should be used for the preparation of LipoRNAi™ Transfection Reagent/sRNA complexes.e. Insufficient incubation time after transfection, which may be misinterpreted as low transfection efficiency. The incubation time required for significant expression in different cells after transfection is usually around 48 hours.f. Regularly check cells for mycoplasma infection. Mycoplasma contamination detrimentally affects cell proliferation, and thereby the transformation efficiency.g. Use cells with less than 30 passages. Excessive passaging affects transfection efficiency to some extent.h. If the expression level of the target protein is not reduced, check the sequence of the transfected sRNA to ensure that its open reading frame is correct.i. If the knockdown of target genes is not effective, design a different siRNA.2. Cellular toxicity occurs:a. Cells should be plated for at least 18-24 hours prior to transfection. b. Reduce the amount of siRNA and also LipoRNAi™ Transfection Reagent proportionally. c. Make sure cell density is not too low at the time of transfection. d. Check cells for mycoplasma or other microorganism infection.e. Change new culture medium 4-6 hours after transfection.Appendix:The size, culture area, cell numbers and recommended culture volume for commonly used multi-well plates and petri dishes:Multiple Well Plates or DishesSingle Well Only for PlatesDiameter(Bottom, mm)*Growth Area(cm2)*AverageCell YieldTotal WellVolume (ml)WorkingVolume (ml)Recommended Volume (ml)6 well34.89.59.5 × 10516.81.9-2.9212 well22.13.83.8 × 1056.90.76-1.14124 well15.61.91.9× 105 3.40.38-0.570.548 well11.00.959.5× 104 1.60.19-0.2850.2596 well6.40.323.2 × 1040.360.10-0.200.1384 well2.70.0565.6 × 1030.1120.025-0.0500.0301536 well1.63 × 1.63**0.0252.5 × 1030.1250.005-0.0100.0103.5 cm dish3499.0 × 105NA1.8-2.726 cm dish52212.1 × 106NA4.2-6.3510 cm dish8.4555.5 × 106NA11-16.51215cm dish141521.5 × 107NA30.4-45.63524.5cm dish22.4 × 22.4**5005.0 × 107NA100-150120*Diameter and growth area may vary depending on the manufacturer, and the listed sizes are from Corning. **These wells or dishes are square.
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