1) For each well of cells, dilute 8.4 ng of siRNA (0.6 pmoles) with 100 μL of serum-free medium (such as OPTI-MEM I medium) and mix well.

2) Immediately add 2 μL of transfection reagent to 100 μL of siRNA, vortex for 10 seconds, and mix well.

3) Incubate at room temperature for 10 min to form the siRNA-PEI cationic nucleic acid transfection reagent complex.

[Note] The incubation time should not exceed 30min

4) During complex formation, remove the cell growth medium and add 500 μL of fresh prewarmed complete medium to each well.

5) Add 100 µL of siRNA-PEI cationic nucleic acid transfection reagent complex directly into the cells, shake the culture plate, and mix gently. The final volume is 600 µL and the final siRNA concentration is 1 nM.

6) Cultivate at 37°C, 5% CO2 incubator until the target gene is expressed. It is suggested that the general mRNA expression level is usually 24-72 h, and the protein expression level is 48-96 h.

Figure 1. Procedure for transfection of adherent cells

[Note]: The transfection efficiency of siRNA will be affected by factors such as target gene, cell type, siRNA titer, the half-life of target mRNA, and the turnover rate of the target protein. The recommended siRNA concentration is between 1 nM and 10 nM. The volume of transfection reagent should be adjusted according to the siRNA concentration and the size of the petri dish, see Table 1 below.

Table 1. When the final concentration of siRNA is 1 nM, the reference value of the dosage of transfection reagent and medium

Culture vessel	Surf. area per well 1 (cm2)	siRNA (pmoles)	Amount of siRNA per well(ng)	Transfection reagent(μL)	Vol. of dilution medium 2(μL)	Vol. of plating medium
96-well	0.3	0.17	2.4	0.7-0.8	50	175 μL
24-well	1.9	0.6	8.6	1-3	100	600 μL
12-well	3.8	1.2	17	2-6	200	1.2 mL
6-well	10	2.2	31	4-12	200	2.2 mL
Flask 25cm2	21	4.4	62	10-20	400	4.4 mL
Flask 75cm2	58	10.5	147	30-50	500	10.5 mL

1. The surface area of cell culture plates provided by different manufacturers may vary;

2. Volume of the medium used to dilute complexation.

Table 2. When the final concentration of siRNA is 10 - 50 nM, the reference value of the dosage of transfection reagent and medium

Culture vessel	Surf. area per well 1 (cm2)	Transfection reagent(μL)	Vol. of dilution medium 2(μL)	Vol. of plating medium
96-well	0.3	0.5-1.5	50	175 μL
24-well	1.9	2-4	100	600 μL
12-well	3.8	4-6	200	1.2 mL
6-well	10	8-16	200	2.2 mL
Flask 25cm2	21	15-25	400	4.4 mL

1. The surface area of cell culture plates provided by different manufacturers may vary;

2. Volume of the medium used to dilute complexation.

2 Suspension cell transfection (take 24-well plate and transfection of 5 nM siRNA as an example, please refer to Table 4 for the loading volume of other culture plates)

seeded cells

2.1 To optimize transfection conditions for suspension cells, the volume of medium for suspension cells needs to be reduced compared to adherent cells. Depending on the dish size and the volume of the complete medium, the recommended number of cells to seed in suspension is shown in Table 3 below.

Table 3. On the day of transfection, according to the size of the culture dish, the reference value of the number of cells inoculated in the suspension

Culture vessel	Surf. area per well 1 (cm2)	Vol. of cell (μL)	Cell number
384-well	0.1	25	5×103-1×104
96-well	0.3	50	1×104-2×104
24-well	1.9	200	1×105-2×105
12-well	3.8	500	2×105-4×105
6-well	10	1000	5×105-2×106
Flask 25cm2	21	2000	2×106-5×106

1. The surface area of cell culture plates provided by different manufacturers may vary;

2.2 Prepare the siRNA-PEI cationic nucleic acid transfection reagent complex according to the following system:

1) For each well of cells, dilute 21 ng siRNA (1.5 pmols) with 100 μL of serum-free medium (such as OPTI-MEM I medium) and mix well.

2) Add 4 μL of transfection reagent to 100 μL of siRNA, vortex immediately for 10 seconds, and mix well.

3) Incubate for 15 min at room temperature to form the siRNA-PEI cationic nucleic acid transfection reagent complex.

[Note] The incubation time should not exceed 30 min

4) Add 100 μL of siRNA-PEI cationic nucleic acid transfection reagent complex to 200 μL of cell suspension in each well, and mix gently. The final volume is 300 µL, and the final siRNA concentration is 5 nM.

5) After culturing for 4-6 hours in a 37 ℃, 5% CO2 incubator, add 700 µL of complete medium, and gently shake the culture plate to mix.

6) Continue to incubate in the incubator until the target gene is expressed. It is suggested that the general mRNA expression level is usually 24-72 h, and the protein expression level is 48-96 h.