Cell Transfection | BMM (Bone marrow-derived macrophage) Cells Transfection – saRNA

Reagents List

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

BMM-saRNA 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, detach cells using trypsin, resuspend, and perform cell counting.
• Seed cells at an appropriate density into a 6-well plate 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:

• saRNA: Use high-quality, purified saRNA.
• 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 saRNA and transfection reagent.

II. Preparation of Transfection Complexes

1. Dilute saRNA:

In a sterile microcentrifuge tube, add 125  μL of serum-free culture medium, followed by 2.5 μg saRNA 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 5  μL of Booster Transfection Reagent. Mix gently by pipetting.

3. Complex Formation:

Add the diluted saRNA 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 saRNA–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 incubated saRNA-transfection reagent complexes (total volume approx. 250 μL) dropwise and evenly to the cell culture medium, and gently rock the culture plate to mix.

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: Incubate for an additional 24–72 hours, then assess transfection efficiency or function according to the experimental design (e.g., fluorescent protein expression, mRNA or protein level 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 saRNA and transfection reagent in Opti-MEM rather than DMEM.

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

Experimental Results Analysis

saRNA was transfected into primary bone marrow-derived macrophages using Yeasen Booster, and the knockdown efficiency of the target gene was evaluated by Western blotting.

Figure 1. saRNA transfection in BMM cells using Yeasen Booster, the results demonstrated that Yeasen Booster facilitated highly efficient transfection and significantly enhanced the expression of the target protein.

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.