Reagents List

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

THP-1-siRNA Transfection Protocol

Cell Culture and Passaging (T25 Flask)

• Cell Counting: Remove the cells from the incubator, gently pipette the cell suspension to ensure homogeneity, and use an automated cell counter to determine both cell viability and cell density.
• Cell Passaging (Direct Dilution Method): Based on the cell count, dilute the cells directly into fresh medium to a target density of approximately 5 × 10⁵ cells/mL.
• Cell Passaging (Partial Medium Exchange Method): If the culture supernatant appears yellow (indicating acidification), carefully remove the plate from the incubator and keep it undisturbed to allow cells to settle. Gently tilt the plate so that cells sediment to the bottom, then aspirate the old supernatant using a pipette. Replace with fresh pre-warmed complete medium, followed by gentle resuspension and counting for passaging.
• Cell Passaging (Centrifugation-Based Medium Exchange Method): If cell health is compromised, passage cells via low-speed centrifugation. Transfer the cell suspension into a sterile centrifuge tube and centrifuge at 200 × g for 3–5 minutes. Carefully discard the supernatant, resuspend the cell pellet in fresh complete medium, and proceed with cell counting and replating at the desired density.

Cell Transfection (6-well-plate)

I. Preparation

1. Cell Preparation: 

• 24 hours before transfection, detach cells using trypsin, resuspend, and perform cell counting. 
• Seed cells at an appropriate density into a 6-well plate or T25 culture flask with 2 mL complete growth medium per well/flask, respectively.
• The target confluency at the time of transfection should be 60%–80%.

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:

In a sterile microcentrifuge tube, add 125  μL of serum-free culture medium, followed by the appropriate volume of 20 μM siRNA stock to achieve a final siRNA concentration of 50 nM in the transfection mix. Gently pipette to mix thoroughly.

2. Dilute Transfection Reagent:

In a separate sterile tube, combine 125  μL of serum-free culture medium with 10  μL of Booster Transfection Reagent. Mix gently by pipetting.

3. 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: Add the siRNA–Booster complexes dropwise and evenly to the cells (total volume ~250 μL for 6-well; ~500 μL for T25). Gently rock the plate/flask to ensure uniform distribution.

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 (6-well) or 6 mL (T25) fresh, pre-warmed complete medium. If cells appear healthy, medium change is not required.

2. Analysis: Continue culturing for 24–72 hours post-transfection.

Evaluate transfection efficiency and gene knockdown by measuring fluorescent protein expression, mRNA levels (qPCR), or protein expression (Western blot) according to your experimental design.

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

THP-1 human monocytic leukemia cells were transfected with siRNA using Yeasen Booster Transfection Reagent. Transfection efficiency was evaluated by fluorescence microscopy. 

Figure 1. siRNA transfection in THP-1 cells using Yeasen Booster, showing strong and uniform fluorescence indicative of high delivery efficiency.

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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