RNA interference (RNAi) is one of the most significant discoveries in the field of life sciences in recent years. It was rated as one of the top ten scientific advances of 2001 by Science magazine and ranked first among the top ten scientific advances of 2002. It refers to the phenomenon where small double-stranded RNA can specifically degrade homologous mRNA, thereby inhibiting or silencing the expression of specific genes. As long as people know the disease-causing gene of a certain disease, they can design small interfering RNA (siRNA) targeting the mRNA of that gene to inhibit or block the expression of the disease-causing gene, thereby achieving the purpose of treating the disease. Since RNAi technology can specifically knock out or silence the expression of specific genes, it has been widely used in exploring gene functions and gene therapy fields such as infectious diseases and malignant tumors. RNAi is currently the most popular research field in life sciences and also the most promising field for new drug development in the future. In addition, RNAi technology can be widely applied in various fields such as agriculture, forestry, animal husbandry, and fishery for improved variety cultivation, variety screening, and disease treatment, etc. There are several methods for introducing siRNA/miRNA into cells: chemical transfection technology, electroporation, calcium phosphate co-precipitation technology, microinjection, and vector introduction technology. Since electroporation causes relatively great damage to cells, it is generally not recommended. Chemical transfection technology is the most commonly used method at present, and the key to the success of chemical transfection lies in the selection of transfection reagents.
siRNA/miRNA Transfection Reagent (M1505992) is a new type of transfection reagent based on cationic polymer, which is suitable for the transfection of siRNA and miRNA. Its principle is that positively charged polymers form positively charged complexes with the negatively charged phosphate groups of siRNA/miRNA. These complexes then interact with negatively charged proteoglycans on the cell surface and enter the cell through endocytosis to form endosomes. This transfection reagent has a proton sponge effect, which can absorb H⁺ in lysosomes. The continuous inflow of protons causes the complex to swell and rupture, thereby releasing exogenous siRNA or miRNA into the cytoplasm and achieving cellular transfection of siRNA/miRNA.
siRNA/miRNA Transfection Reagent (M1505992) has high transfection efficiency in various common cells, with advantages such as high efficiency, low toxicity, simple operation, and good reproducibility. It is suitable for siRNA/miRNA-mediated gene silencing experiments. Its unique formula results in no obvious cytotoxicity after transfection, so there is no need to remove the transfection reagent-nucleic acid complex or replace it with fresh medium, and the transfection system can also be optimized according to specific conditions.
Product Features:
⁕ Excellent transfection efficiency — It exhibits outstanding transfection efficiency across a wide range of cell types, with good gene silencing effects and low off-target effects.
⁕ Low cytotoxicity — It acts gently and can well balance high transfection efficiency and low cytotoxicity.
⁕ Broad application range — Suitable for multiple cell types and applicable to siRNA/miRNA-mediated gene silencing experiments.
⁕ Simple operation — The experimental protocol is simple and fast, enabling stable results. There is no need to remove the complex or replace with fresh medium after transfection.
Scope of Application:
siRNA/miRNA Transfection Reagent is a novel transfection reagent based on cationic polymer, suitable for the transfection of siRNA and miRNA.
Summary of Experimental Procedure:
Figure 1. Flowchart of transfection experiment using siRNA/miRNA Transfection Reagent (M1505992)
Experimental Procedures:
(Take a 24-well plate as an example; for the sample addition volume of other culture plates, refer to Table 1: Transfection Quantity Standards)
1. Prepare Cells for Transfection
(1) Adherent cells: One day before transfection, seed the trypsin-digested cells at a density of 0.1–1×10⁵ cells per well, ensuring the cell density reaches 30%–40% at the time of transfection.
(2) Suspension cells: On the day of transfection, before preparing the transfection reagent-nucleic acid complex, seed the cells in a 24-well plate by adding 0.5–2.5×10⁵ cells to 500 µL of growth medium per well.
⁕ Cell status significantly affects transfection efficiency. Cells to be transfected should be in good growth condition. It is recommended to use cells in the exponential growth phase with a survival rate >90% for transfection.
2. Prepare Transfection Reagent-Nucleic Acid Complex
(1) Add 1 μL of siRNA (20 μM, dissolved in DEPC water) to a 1.5 mL centrifuge tube, then add 2 μL of transfection reagent to the siRNA and mix. Incubate at room temperature for 3 minutes.
⁕ Factors such as gene properties, cell type, siRNA potency, target mRNA half-life, and target protein turnover rate can affect siRNA transfection efficiency. It is recommended to select an siRNA working concentration between 10–100 nM. The volume of transfection reagent should be adjusted according to the siRNA concentration and the size of the culture vessel; please refer to Table 1.
(2) Add 100 μL of Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393) to the above mixture, mix gently, and let stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
3. siRNA Cell Transfection
After 30 minutes, add the 100 µL transfection reagent-nucleic acid complex to each well of cells. Gently swirl the plate to mix, then return the cells to the incubator for continued culture.
4. Analyze Gene Interference Efficiency in Transfected Cells
After culturing the transfected cells for 24–48 hours, the gene interference effect can be detected by methods such as RT-PCR, Western Blot, ELISA, fluorescence detection, flow cytometry, or reporter gene assays, depending on actual needs. Alternatively, proceed with subsequent functional experiments.
⁕ This transfection reagent is also suitable for miRNA transfection; please refer to the siRNA protocol for specific operations.
Table 1: Transfection Volume Standards
Specifications of Cell Culture Equipment | Culture System (mL) | SiRNA(20μM) (μL) | Transfection Reagent (μL) | Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393) |
96-well plate
| 0.1 | 0.2 | 0.4 | 20 |
24-well plate
| 0.5 | 1 | 2 | 100 |
12-well plate
| 1 | 2 | 4 | 200 |
6-well plate
| 2 | 4 | 8 | 400 |
60mm Dish
| 4 | 8 | 16 | 800 |
100mm Dish
| 10 | 20 | 40 | 2000 |
Precautions:
1. Nucleic Acid Quality: Ensure that the siRNA/miRNA has undergone PAGE purification and desalination. High-purity siRNA/miRNA helps achieve high transfection efficiency.
2. Cell Quality: Cell status significantly affects transfection efficiency. It is recommended to use cells in the exponential growth phase with a survival rate >90% for transfection.
3. Cell Density: Transfection is recommended to be performed within 12–24 hours after cell passage, when the cell density is 30%–40%. Different cell transfection experiments have different requirements for cell density. When transfecting different nucleic acids or different cell lines, it is necessary to re-optimize the experimental conditions according to the instruction manual. In addition, ensure consistent seeding conditions during the experiment to guarantee the reproducibility of experimental data.
4. Ratio of siRNA to Transfection Reagent: For most cell lines, the ratio of siRNA (μL of 20 μM Stock) to transfection reagent (μL) in the transfection complex ranges from 1:1 to 1:3, with a recommended ratio of 1:2. To obtain optimal gene silencing results, this ratio needs to be optimized, and a suitable transfection ratio should be selected based on the transfected cells and siRNA.
5. Since certain components in some special media may inhibit cationic polymer-mediated transfection, it is necessary to test the compatibility between the special medium and this product.
6. Before transfection, ensure that the silencing of siRNA/miRNA target gene expression does not affect cell viability.
7. You need to design several siRNAs (small interfering RNAs) targeting the gene of interest and evaluate their potency.
8. Use RNase-free and pyrogen-free materials (such as centrifuge tubes, pipette tips, and buffers) throughout the experiment.
9. For your health and safety, please operate in a standardized manner and wear a lab coat and gloves during the experiment.
10. This product is for research use only and shall not be used for clinical diagnosis or treatment.
Experimental Case Analysis:
1. One day before transfection, HeLa cells stably expressing GFP were seeded into a 24-well plate, ensuring the cell density reached 30% at the time of transfection.
2. For each well, add 1 μL of siRNA (GFP/NC, 20 μM) to a 1.5 mL centrifuge tube, then add 2 μL of transfection reagent (M1505992) to mix with the siRNA, and incubate at room temperature for 3 minutes.
3. Add 100 μL of Optimal-MEM Reduced Serum Medium (with Phenol Red) (Cat. No. O778393) to the above mixture, mix gently, and let it stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
4. After 30 minutes, add the 100 µL transfection reagent-nucleic acid complex to each well of cells, gently swirl the plate to mix, and place it in the incubator for continued culture.
5. Twenty-four hours after siRNA transfection, the interference effect on GFP (Green Fluorescent Protein) was detected using a fluorescence microscope and flow cytometry. The experimental results are shown in Figure 2.
Figure 2. GFP interference effect detected by fluorescence microscopy (A) and flow cytometry (B) at 24 hours after transfection with GFP siRNA using Lentivirus Packaging Transfection Reagent (S1505995) and Thermo Fisher RNAiMAX.
Note: In this experiment, the commercial siRNA transfection reagent RNAiMAX was selected as the control group, and the specific experimental operation of RNAiMAX was performed according to its instruction manual. Briefly: Dilute 1 μL of siRNA (GFP, 20 μM) in 50 μL of serum-free Opti-MEM medium without antibiotics and mix gently. Then, dilute 1 μL of RNAiMAX in 50 μL of serum-free Opti-MEM medium without antibiotics and mix gently. Gently mix the diluted siRNA with the diluted RNAiMAX and incubate at room temperature for 20 minutes. Add the mixture to the cells to a final volume of 600 μL. At 24 hours after siRNA transfection, the interference effect on GFP (Green Fluorescent Protein) was detected using a fluorescence microscope and flow cytometry.
ZJ25F1230401