1. Experimental Materials
- HEK 293 cell: Cell confluency should reach 70%–90%.
- mRNA: EGFP mRNA, 1000 bp (Concentration ≥ 100 ng/μL)
- Hieff TransTM Booster DNA&RNA Transfection Reagent (Cat#40801)
- Serum-free Opti-MEM® (Reduced Serum Medium)
- Compatible tissue culture plates and related lab consumables (using a 6-well plate as an example)
2. Experimental Procedure
1)Preparation One Day Before Transfection
- Seed HEK293 cells in a 6-well plate at a density that will reach 70–90% confluency on the day of transfection.
- Use 2 mL complete DMEM (with 10% FBS) per well.
- Incubate at 37 °C, 5% CO₂ overnight
2)Prepare mRNA–Reagent Complex(per well of a 6-well plate)
- Dilute mRNA: Dilute 2.5 μg EGFP mRNA in 100 μL Opti-MEM.
[Note]: mRNA concentration should be > 0.1 μg/μL to ensure accurate pipetting.
- Dilute Transfection Reagent: In a separate tube, dilute 5 μL transfection reagent in 100 μL Opti-MEM.
- Form mRNA-Transfection Reagent Complex: Combine the two solutions, mix gently, and incubate for 10–15 minutes at room temperature.
|
Plate Format |
mRNA Amount per Well |
Transfection Reagent Volume |
Total Complex Volume (in Opti-MEM or serum-free medium) |
|
6-well plate |
2–3 μg |
4–6 μL |
200 μL |
|
24-well plate |
500–800 ng |
1.5–2 μL |
50 μL |
|
96-well plate |
100–200 ng |
0.3–0.5 μL |
20 μL |
3)Cell Transfection: Add to Cells
- Remove old culture medium from the well and replace with 1.8 mL fresh complete DMEM.
- Add the 200 μL mRNA–reagent complex dropwise to the well.
- Gently rock the plate to distribute evenly.
4)Post-Transfection Handling
- Incubate cells at 37 °C, 5% CO₂.
- Observe EGFP expression at 18–24 hours post-transfection using fluorescence microscopy.
- No media change is needed unless cytotoxicity is observed.
Tips & Notes
- Ensure mRNA is of high purity (DNase-treated, A260/A280 ~2.0).
- Avoid repeated freeze-thaw cycles of mRNA stock.
- This protocol can be adapted for other plate formats (e.g., 24-well or 96-well) by scaling volumes accordingly.
3. Experimental Result
1）Efficient mRNA Delivery in HEK293 cell
Figure 1. Transfection of 2.5 μg EGFP mRNA (~1000 bp) with 5 μL transfection reagent in a 6-well plate, showing high transfection efficiency.
2）Efficient mRNA Delivery in a Wide Range of Cell Types
Figure 2. mRNA Transfection Performance in Multiple Cell Lines
mRNA transfection across different cells using Booster DNA&RNA Transfection Reagent versus T brand L*3000. The result demonstrates superior mRNA transfection efficiency of Booster.
4. Customer Feedback
1) High-Efficiency 293T mRNA Transfection
Figure 3. mRNA Transfection Performance
2) High-Efficiency HTC116 mRNA Transfection
Figure 4. mRNA transfection using Booster DNA&RNA Transfection Reagent versus T brand L*3000. The result demonstrates superior mRNA transfection efficiency of Booster.
4. Frequently Asked Questions (FAQs) & Solutions
Q1: What is the optimal confluency for mRNA transfection?
A: Cells should be 70%–90% confluent at the time of transfection to ensure optimal uptake and cell health.
Q2: Can I use serum-containing medium during transfection?
A: Most polymer-based transfection reagents, including Hieff Trans™ Booster, are serum-compatible. However, avoid serum during complex formation; use serum-free medium (e.g., Opti-MEM) for that step.
Q3: What concentration should my mRNA stock be?
A: The mRNA concentration should be at least 100 ng/μL. For best results, use ≥ 0.5 μg/μL to minimize pipetting error.
Q4: How much mRNA should I use per well?
A: For a 6-well plate, we recommend 2–3 μg mRNA per well. Adjust accordingly for other formats (e.g., ~500–800 ng for 24-well).
Q5: How soon can I detect protein expression after transfection?
A: EGFP expression can typically be observed 6–12 hours post-transfection, with peak expression at 24–48 hours.
Q6: What should I do if transfection efficiency is low?
A: Ensure cells are healthy and at proper confluency.
- Confirm mRNA quality: A260/A280 ~2.0, intact RNA (no degradation), and the presence of both a 5' cap and a 3' poly(A) tail. Increase the amount of mRNA used within a reasonable range.
- Optimize the mRNA/reagent ratio: Adjust the mRNA/reagent ratio between 1:3 and 1:5 for optimal performance.
- Control the incubation time of the complex: Incubate the complex for 5–10 minutes to avoid aggregation."
- Optimize the detection time: Expression peaks may vary by cell type—commonly within 6–24 hours, but extended detection up to 48 hours may be required.
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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