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 culture medium, add 3–4 mL of room-temperature PBS, gently shake 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 cell gaps widen and cells become rounded, gently pipette with trypsin to dislodge the cells.
• Termination: Add 2–3 mL of complete culture medium to stop digestion, then pipette to mix and form a uniform cell suspension.
• Seeding: Transfer the suspension to a centrifuge tube, centrifuge at 200 ×g for 3–5 minutes, discard the supernatant, resuspend the cell pellet in fresh culture medium, seed into new culture flasks, add appropriate volume of medium, and continue incubation.

Cell Transfection (6-Well Plate)

I. Preparation

1. Cell Preparation: 24 hours before transfection, digest cells with trypsin, resuspend, and perform cell counting. Seed cells into a 6-well plate, adding 2 mL of complete medium per well. The goal is to achieve 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.

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. Complex Formation

1. Dilute siRNA: In a sterile centrifuge tube, add 150 μL of serum-free medium, followed by 5 μL of siRNA (20 μM). Mix gently to obtain diluted siRNA solution.

2. Dilute Transfection Reagent: In another sterile centrifuge tube, add 125 μL of serum-free medium, followed by 5μL(or 7.5 μL) of Hieff Trans™Booster transfection reagent. Gently pipette to mix.

3. Mix and Incubate: Add the diluted siRNA solution to the tube containing the diluted transfection reagent. 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 Change: Before transfection, carefully aspirate the old medium and replace with 2 mL pre-warmed complete medium.  

2. Add Complexes: Add the incubated siRNA-transfection reagent complexes (~250 μL total) dropwise and evenly to the cells. Gently rock the plate to distribute.  

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, check cell morphology. If significant toxicity is observed, aspirate the medium containing complexes and replace with 2 mL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional.  

2. Analysis: After 24–72 hours, assess transfection efficiency or functional outcomes based on experimental design (e.g., fluorescence observation, mRNA or protein detection).

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

Transfection of siRNA into RAW264.7 mouse monocytic macrophage cells using Yeasen Booster DNA/RNA Transfection Reagent, followed by qPCR analysis to evaluate transfection efficiency.

Figure 1. siRNA transfection in RAW264.7 cells using Yeasen Booster DNA&RNA Transfection Reagent versus L*3000. The result demonstrates that  Booster achieves a stronger gene knockdown effect.

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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