Reagents List
|
Product Name |
Cat.NO. |
|
Hieff Trans™ RNAiBoost Transfection Reagent (RNAiMax alternative) |
40807ES |
H9c2 siRNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Washing: The old culture medium was aspirated. 3–4 mL of room temperature PBS was added to gently rinse the flask for 10–20 seconds, and then aspirated.
- Digestion: 1 mL of trypsin was added to wet the bottom of the flask, which was then incubated at 37°C for digestion.
- Detachment: When the intercellular spaces widened and the cells became rounded, the cells were gently dislodged by pipetting with the trypsin.
- Termination: 2–3 mL of complete medium was added to terminate the digestion. The mixture was pipetted to form a uniform cell suspension.
- Seeding: The suspension was collected and centrifuged at 200 ×g for 3–5 minutes. The supernatant was discarded, and the cell pellet was resuspended in fresh medium. The cells were then subcultured into new flasks, topped up with fresh medium, and returned to the incubator for continued culture.
H9c2 cells were routinely cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified incubator at 37°C with 5% CO2. The cells were passaged for subsequent transfection experiments upon reaching 85%–90% confluence.
Cell Transfection (24-Well Plate)
I. Preparation
1. Cell Preparation: 24 hours before transfection, the cells were harvested via trypsinization and resuspended for cell counting. The cells were then seeded into a 24-well plate at an appropriate density (4 × 10⁴ cells per well) with 500 μL of complete medium added to each well. The target confluence at the time of transfection was 60%–80%. In this experiment, transfection was initiated when the cell confluence reached 50%–60%.
2. Reagent Preparation:
- siRNA: Synthesize high-quality siRNAs. Typically, three different siRNAs are designed for validation, with the initial concentration adjusted to 20 μM.
- Transfection Reagent: Allow Hieff Trans™ RNAiBoost Transfection Reagent to equilibrate to room temperature before use. Mix gently by pipetting.
- Serum-free Medium: Prepare a serum-free base medium (e.g., Opti-MEM) for diluting siRNA and transfection reagent.
II. Cell Transfection Protocol
1. Dilute siRNA: Pipette 3 μL of siRNA solution and mix thoroughly with 15 μL of Enhancer solution by pipetting up and down.
2. Dilute Transfection Reagent: Add 6 μL of RNAiBoost in vitro transfection reagent to the mixture from step (1). Mix thoroughly by pipetting and incubate at room temperature for 8 minutes.
3. Cell Transfection: After incubation, add 15 μL of Opti-MEM transfection diluent to the mixture from step (2) and mix well. Add the resulting 25 μL transfection complex to one well of a 24-well plate. Gently rock the plate to ensure even distribution. The complex can be scaled up according to the number of wells required.
4. Cell Culture and Detection: Return the cells to a 37°C, 5% CO₂ incubator. Assess the gene silencing efficiency 24–48 hours post-transfection.
III. Post-Transfection Handling
1. Medium Replacement: 4–6 hours post-transfection, check cell morphology. If significant toxicity is observed, aspirate the medium containing complexes and replace with 500 μL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional.
2. Analysis: Typically, RNA is extracted for analysis 48 hours after transfection. In this experiment, WB was performed 48 hours post-transfection.
Tips:
1. If significant cytotoxicity is observed after transfection, consider replacing the medium at 6 hours or halving the reagent dosage to avoid cytotoxicity issues.
2. If the transfection efficiency is relatively low, increasing the amount of transfection reagent can effectively improve it.
3. Opti-MEM is recommended for diluting siRNA and transfection reagents.
4. The transfection reagent is compatible with serum and antibiotics; however, the medium used to dilute the plasmid and transfection reagent must be serum-free and antibiotic-free.
5. Strictly adhere to the recommended dosages in the table for preparing the transfection complex. If you need to increase or decrease the dosage, do so proportionally. For example, for a 6-well plate, if the final concentration is 50 nM, prepare 100 µL of transfection complex according to the manual. If you need to double the dosage, prepare 200 µL of transfection complex and add it to the well. Conversely, if you halve the dosage, simply add 50 µL of the prepared 100 µL transfection complex to the well.
Experimental Results Analysis
In this study, H9c2 rat cardiomyocytes were transfected with siRNA using Yeasen RNAiBoost Transfection Reagent, and the transfection efficiency was subsequently evaluated by Western Blot (WB).
Figure 1. siRNA transfection inH9c2 Rat Cardiomyocytes cell was performed using Yeasen RNAiBoost. The results demonstrated that Yeasen RNAiBoost achieved highly efficient transfection, leading to a significant downregulation of the target gene expression. Notably, the reagent exhibited minimal cytotoxicity towards H9c2 cells. Post-transfection, the cells maintained excellent morphology with very few dead cells observed. The transfection efficiency was both stable and robust, yielding clear and reliable results in subsequent qPCR and WB assays, thus ensuring high experimental reproducibility.
Guidelines for RNAiBoost Reagent Dosage in Different Culture Dishes(for reference only):
|
Culture Plate |
Culture Medium Volume |
siRNA Solution (Stock 20 μM) |
Enhancer |
Transfection Reagent |
Serum-free Medium Volume |
Transfection Complex Volume |
|
24-well plate |
500 μL |
1.25 μL |
6.25 μL |
2.5 μL |
15 μL |
25 μL |
|
12-well plate |
1.0 mL |
2.5 μL |
12.5 μL |
5 μL |
30 μL |
50 μL |
|
6-well plate |
2.0 mL |
5 μL |
25 μL |
10 μL |
60 μL |
100 μL |
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