1. Experimental Materials
- HEK293 cells: Cell confluency should reach 30%–50% at the time of transfection.
- siRNA: Target-specific or control siRNA (Concentration ≥ 10 μM)
- Hieff TransTM Booster DNA&RNA Transfection Reagent (Cat#40801)
- Culture Media: Complete growth medium (containing serum and antibiotics)
- Serum-free medium (e.g., Opti-MEM® I Reduced Serum Medium)
[Note]: Serum-free medium is used to dilute siRNA and transfection reagent for complex preparation.
Other Materials: Sterile centrifuge tubes, Micropipettes and sterile pipette tips, CO₂ incubator, Centrifuge.
2. Experimental Procedure
1)Preparation One Day Before Transfection
- Seed HEK293 cells in a 24-well plate at a density that will reach 30%–50% confluency on the day of transfection.
- Use 0.5-1 mL complete DMEM (with 10% FBS) per well.
- Incubate at 37 °C, 5% CO₂ overnight.
[Note]: For a 24-well plate, seed 0.5–1 mL cell suspension per well. Gently rock the plate to distribute cells evenly.
2)Prepare siRNA–Reagent Complex(per well of a 6-well plate)
- Dilute siRNA: Dilute 50 nM final concentration of siRNA in 50 μL Opti-MEM.
[Note]: siRNA stock is typically prepared at 10–20 μM.
- Dilute Transfection Reagent: In a separate tube, dilute 1.5-2 μL transfection reagent in 50 μL Opti-MEM.
- Form siRNA-Transfection Reagent Complex: Combine the two solutions, mix gently, and incubate for 10–15 minutes at room temperature.
|
Plate Format |
Final siRNA per Well |
Transfection Reagent Volume |
Total Complex Volume (Opti-MEM or serum-free medium) |
|
6-well plate |
50–100 nM |
4–6 μL |
200 μL |
|
24-well plate |
10–50 nM |
1.5–2 μL |
50 μL |
|
96-well plate |
10–30 nM |
0.3–0.5 μL |
20 μL |
3)Cell Transfection: Add to Cells
- Remove old culture medium from the well and replace with 0.5-1 mL fresh complete DMEM( (serum-containing, no antibiotics)..
- Add the 100 μL siRNA–reagent complex dropwise to the well.
- Gently rock the plate to distribute evenly.
4)Post-Transfection Handling
- Incubate cells at 37 °C, 5% CO₂.
- Analyze gene knockdown 24–72 hours post-transfection, depending on the target gene and cell type.
- No medium change is necessary unless cytotoxicity is observed.
[Note]: Time points vary by target; typically, qPCR at 24–48 h, protein knockdown at 48–72 h post-transfection.
3. Experimental Result
1. Efficient siRNA Delivery in a Wide Range of Cell Types
Figure 1. siRNA transfection across different cells using Booster DNA&RNA Transfection Reagent versus T brand L*3000. The result demonstrates superior siRNA transfection efficiency of Booster.
4. Frequently Asked Questions (FAQs) & Solutions
Q1: What is the optimal confluency for siRNA transfection?
A: Cells should be at 30%–50% confluency at the time of transfection to ensure efficient uptake and maintain cell viability.
Q2: Can I use serum-containing medium during transfection?
A: Most polymer-based transfection reagents, including Hieff Trans™ siRNA Reagent, are compatible with serum. However, use serum-free medium (e.g., Opti-MEM) during complex formation to ensure optimal complexation.
Q3: What concentration should my siRNA stock be?
A: siRNA stock concentrations typically range from 10 to 20 μM. Dilute appropriately to reach a final concentration of 10–50 nM in the transfection reaction.
Q4: How much siRNA should I use per well?
A: For a 24-well plate, a final siRNA concentration of 10–50 nM per well is recommended. Adjust volumes accordingly for other plate formats.
Q5: When can I detect gene knockdown after transfection?
A: mRNA knockdown can typically be detected at 24–48 hours post-transfection; protein knockdown is often observed between 48–72 hours.
Q6: What should I do if transfection efficiency is low?
A:
- Ensure cells are healthy and at the recommended confluency (30%–50%).
- Verify siRNA integrity and sequence specificity; avoid degraded siRNA.
- Optimize the siRNA-to-reagent ratio within recommended ranges (e.g., 10–50 nM siRNA with 1.5–2 μL reagent per well in 24-well format).
- Control the incubation time of the siRNA–reagent complex: incubate for 10–15 minutes at room temperature to form stable complexes.
- Optimize detection time, as knockdown kinetics vary by cell type and gene target.
Note: Use fresh complete medium after transfection and avoid antibiotics during the transfection period to minimize cytotoxicity.
5. Related Products
|
Application |
Name |
Catalog No. |
Size |
|
Works with DNA, siRNA, miRNA, mRNA, and ASO, etc. Proven success in primary cells. |
40801 |
100 μL/1.5 mL |
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