T&L Biotechnology

Product Name

Generic Name

Lipidnano^TM Super RNAi Transfection Reagent

Product Information

Storage Conditions: Transport with ice packs, store at 4ºC (do not freeze)

Expiry Date: 12 months

Product Description

Lipidnano^TM Super RNAi Transfection Reagent is a highly efficient, non-strongly positive type lipid transfection reagent, which is used for siRNA-mediated gene knockdown experiments. And it is suitable for efficient delivery of small RNAs such as siRNA and miRNA into a wide range of cell types with unique ultra-high cell utilization rate. The reagent has excellent nucleic acid loading efficiency, and promotes RNA-interfering function of siRNA in the cytoplasm by improving the escape level of endosome after endocytosis. This process of action is highly broad-spectrum. An efficient target gene knockdown level can be achieved in a low concentration range, with low cytotoxicity. Furthermore, this reagent is not affected by serum and antibiotics.

Directions For Use 

In vitro transfection protocol:

1. Seeding cells: It is recommended to seed cells a day in advance. For the number of seeded cells, refer to the recommended amount below.

2. Reagent preparation:

2.1 Preparation of siRNA dilution: SiRNA is diluted to 140 μg/mL with sterile water.

2.2 Preparation of siRNA-transfection complex: Add 2 μL of siRNA dilution and 14 μL of Lipidnano^TM Super RNAi Transfection Reagent Solution A into a sterile centrifuge tube, and mix well. Add 4 μL of Lipidnano^TM Super RNAi Transfection Reagent Solution B, and mix well again. Leave the mixture at room temperature for 5 min, then add 180 μL of culture medium, and mix well once more to complete the preparation of transfection complex.

3. Add the sample: Add the proper amount of prepared siRNA-transfection complex into cell culture plates according to the recommended list below, and gently shake the culture plate to mix well.

4. Cell culture: Culture transfected cells in an incubator at 37 ℃ and 5% CO₂ until the interfering effect is observed. It is recommended to detect the mRNA level at 24-48 h, and protein level at 48–72 h after transfection, respectively.

Table 1. Examples of siRNA transfection

Note: In order to obtain the best gene silencing effect, the optimal range of siRNA dose for each cell line needs to be determined experimentally.

Application Fields:

1. Transfection of adherent and suspension cells

2. Gene transfection of some primary cells and transformed cell lines

3. High-throughput siRNA transfection experiments

Points To Note:

1. Before transfection, it is important to ensure that siRNA is purified through PAGE and desalted, since high purified siRNA or miRNA helps to obtain high transfection efficiency, and it is also important to ensure that post-transcriptional gene silencing does not affect cell viability.

2. First transfection: If you are transfecting your cell line for the first time, it is recommended to try several concentrations of siRNA-transfection complex solution and vary the concentration of siRNA in the range of 0.014 -0.70 μg/mL. Then adjust the amounts of reagent according to the results of the first transfection.

3. Selection of diluent for siRNA-transfection reagent: There is no special requirement for the medium, since complete medium, low-serum and serum-free medium are all available.

4. Concentration conversion: The conversion between molar concentration and mass concentration can be done according to the molecular weight of the siRNA. X nmol/L*molecular weight = Y ng/L, for example, a siRNA with a molecular weight of 14000, 1 nmol/L*14000=14000 ng/L=14 μg/L.

5. Density of cells to be transfected: It is recommended to perform transfection at about 70% of cell confluency. Analysis of gene-silencing results is usually performed after transfection 24 hours. The extremely low cytotoxicity of this transfection reagent enables researchers to select suitable conditions for transfection according to the growth features of target cells and the expression features of target genes. Repeatability of transfection should be ensured via good quality cell cultures and rigorous operations. Generally, the transfection efficiency of healthy cells is higher, and low passage of cells ensure the stability of the cells used in each experiment. Cells within 50 passages is recommended for transfection.

6. Selection of positive control: GADPH-siRNA is a good positive control for most cells. Measure the reduction of target mRNA level relative to untransfected cells using reference genes such as β-actin mRNA as a control after transfecting different concentrations of positive control siRNA or experimental siRNA into target cells about 24 hours.

Traditional transfection reagents enable cells uptake a large number of siRNAs by their surface positive charges. However, after entering the cells, the escape level of siRNA endosome is low, which results in the inability of the uptaken siRNA to work and also causes cytotoxicity. This product is a non-strongly positive type lipid transfection reagent, which avoids the cytotoxicity caused by large amount of reagent uptake. However, this product can improve the escape efficiency of siRNA endosomes after endocytosis, allowing more release of siRNAs into cytoplasm by promoting the escape of siRNA endosomes to achieve efficient gene silencing. Therefore, optimizing method by testing fluorescently labeling siRNA at the cell level is not suitable for this transfection reagent. It is recommended to use western blot or QPCR to optimize the transfection concentration.

7. Operation notes: After mixing siRNA and transfection reagent solution A, add solution B and mix well. Place the mixture for 5 min, then dilute it with culture medium. Finally add the mixture into the cell wells within 1 hour, and shake the culture plate back and forward gently to mix the contents well.

8. Avoiding RNase contamination: Trace amounts of RNase can cause siRNA experiments to fail. RNases are ubiquitous in the experimental environment, such as skin, hair, all objects that have been touched by hand or exposed to air. Therefore, it is important to make sure that each step of the experiment is free of RNase contamination. Commercial enzyme-free laboratory consumables are recommended.

9. Effect evaluation: The efficiency of cell transfection can differ due to variations in the cell and siRNA types. The optimal time to observe the effect of siRNA transfection is 24 to 48 hours after transfection at the mRNA level and 48 to 72 hours after transfection at the protein level. SiRNA interference is nonlinear. The half-life of different genes is quite different. When the interference effect of the siRNA is measured for the first time, multi-point measurement is recommended to determine the best time point.