Reagents List

Product Name	Cat.NO.
Hieff Trans™ Booster DNA/RNA Transfection Reagent	40801ES

RAW264.7-siRNA Transfection Protocol

Cell Culture and Passaging (T25 Flask)

• Washing: Aspirate the old medium, add 3–4 mL of room-temperature PBS, gently swirl to rinse for 10–20 seconds, then aspirate.
• Digestion: Add 1 mL of trypsin to cover the bottom of the flask and incubate at 37°C for digestion.
• Detachment: When cells begin to separate and round up, gently pipette the trypsin up and down to dislodge the cells.
• Termination: Add 2–3 mL of complete medium to inactivate the trypsin, then pipette to resuspend and create a uniform cell suspension.
• Seeding: Transfer the suspension to a centrifuge tube, centrifuge at 200 × g for 3–5 minutes, aspirate the supernatant, resuspend the cell pellet in fresh medium, seed into new culture vessels, and add fresh medium to the required volume for continued culture.

Cell Transfection (6-well-plate)

I. Preparation

1. Cell Preparation: 24 hours before transfection, cells were detached with trypsin, resuspended, and counted. They were then seeded into 6-well plates at an appropriate density (5 × 10⁵ cells/well) with 2 mL of complete growth medium per well, aiming for 60–80% confluency at the time of transfection.

2. Reagent Preparation:

• siRNA: Use high-quality, purified siRNA. It is recommended to design and test at least three siRNA sequences for optimal gene silencing. The siRNA lyophilized powder was reconstituted to a working stock concentration of 20 μM.
• Transfection Reagent: Allow Hieff Trans™Booster 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. Preparation of Transfection Complexes

1. Dilute siRNA:

2. In a sterile microcentrifuge tube, add 125 μL of serum-free medium, followed by 5 μL of siRNA (20 μM). Gently mix to obtain the diluted siRNA solution.

3. Dilute Transfection Reagent:

In a separate sterile tube, combine 125  μL of serum-free culture medium with 7.5  μL of Booster Transfection Reagent. Mix gently by pipetting.

4. Complex Formation:

Add the diluted siRNA solution to the diluted Booster solution. Mix gently by pipetting. Incubate the mixture at room temperature for 10–15 minutes to allow formation of stable siRNA–transfection reagent complexes.

III. Transfection

1. Medium Replacement: Before adding the complexes, carefully remove the old culture medium and replace it with 2 mL fresh, pre-warmed complete medium.

2. Add Complexes: Gently add the pre-incubated siRNA–transfection reagent complex (total volume ~250 μL) dropwise and evenly into the cell culture medium, then gently rock the plate to ensure uniform mixing.

3. Incubation: Return cells to a 37°C, 5% CO₂ incubator for culture.

IV. Post-Transfection Handling

1. Medium Replacement: 4–6 hours post-transfection, carefully examine cell morphology. If significant cytotoxicity is observed, aspirate the medium containing the complexes and replace with 2 mL fresh, pre-warmed complete medium. If cells appear healthy, medium change is not required.

2. Analysis: After an additional 24 hours of incubation, total RNA was extracted for analysis.

Tips:

1. If significant cytotoxicity occurs, replace medium after 6 h or reduce Enhancer volume by half to mitigate toxicity.  

2. If transfection efficiency is low, increasing the amount of transfection reagent may improve results.  

3. For optimal performance, dilute siRNA and transfection reagent in Opti-MEM rather than DMEM.  

4. The transfection system is compatible with serum and antibiotics; however, siRNA and reagent dilutions must be performed in serum- and antibiotic-free medium.

Experimental Results Analysis

siRNA was transfected into RAW264.7 mouse monocyte/macrophage cells using Yeasen Booster Transfection Reagent, and transfection efficiency was evaluated by qPCR.

Figure 1. siRNA transfection in RAW264.7 mouse monocyte/macrophage cells using Yeasen Booster Transfection Reagent. Results show that Yeasen Booster  enables highly efficient siRNA delivery in RAW264.7 cells, leading to significant knockdown of the target gene expression.

Different Cell Culture Vessel Transfection Volumes (for reference only):

Culture vessel	Medium Volume		DNA Transfection			siRNA Transfection (Final Concentration 50 nM)
	Volume of Medium	Volume of Opti-MEM Complex	DNA(μg)	Booster Transfection Reagent (μL)	Transfection Enhancer (μL)	Volume of siRNA (Initial Concentration 20 μM)	Booster Transfection Reagent (μL)
96-well	100 μL	2×5 μL	0.1	0.2	0.2	0.25 μL	0.3
48-well	250 μL	2×12.5 μL	0.25	0.5	0.5	0.625 μL	0.75
24-well	500 μL	2×25 μL	0.5	1	1	1.25 μL	1.5
12-well	1 mL	2×50 μL	1	2	2	2.5 μL	3
6-well	2 mL	2×125 μL	2.5	5	5	5 μL	7.5
60 mm	5 mL	2×250 μL	5-10	10-20	10-20	12.5 μL	20
10 cm	10 mL	2×500 μL	15-25	30-50	30-50	25 μL	40
T25	6 mL	2×250 μL	6-12	12-24	12-24	15 μL	24
T75	15 mL	2×750 μL	20-40	40-80	40-80	37.5 μL	60

[Note]: The volumes provided in this table are for reference only and are suitable for both suspension and adherent cells. For suspension cells, the test can be performed at a cell density of 0.5–1×10⁶ cells/mL. The actual amounts of DNA and Booster DNA/RNA transfection reagent should be optimized depending on cell type and other experimental conditions. It is recommended to maintain a ratio between 1:0.5 and 1:5 (DNA : Booster transfection reagent). The amount and experimental conditions for mRNA are the same as those for DNA. If the cells are particularly fragile and excessive cell death is observed, better results may be achieved by reducing the amount of enhancer by half.

 

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