Reagents List
|
Product Name |
Cat.NO. |
|
40801ES |
GBMs-siRNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Washing: Aspirate old medium and gently rinse with 3–4 mL of room-temperature PBS for 10–20 seconds, then aspirate.
- Digestion: Add 1 mL trypsin to cover the bottom of the flask and incubate at 37°C for digestion.
- Detachment: When cells begin to retract and round up, gently pipette the trypsin solution to dislodge the cells.
- Termination: Add 2–3 mL complete culture medium to stop digestion, and pipette to create a uniform cell suspension.
- Subculturing: Transfer the suspension to a centrifuge tube, centrifuge at 200 × g for 3–5 minutes, discard supernatant, resuspend pellet in fresh medium, seed into new flasks, and add complete medium for continued culture.
Cell Transfection (6-Well Plate)
I. Preparation
1. Cell Preparation:
- 24 hours before transfection, harvest cells using trypsin, resuspend, and count.
- Seed cells into 6-well plates (or other culture vessels) at an appropriate density with 2 mL complete medium per well.
- Target 60–80% confluency at the time of transfection.
2. Reagent Preparation:
- SiRNA: Synthesize high-quality siRNA; typically, three different siRNA sequences are designed for validation.
- Transfection Reagent: Allow LipoBooster 3000 to warm to room temperature before use. Gently mix prior to use.
- Serum-Free Medium: Prepare serum- and antibiotic-free basal medium (e.g., Opti-MEM) for diluting siRNA and transfection reagent.
II. Complex Formation
1. Dilute siRNA: In a sterile microcentrifuge tube, add 125 μL of serum-free medium followed by 5 μg(20 μM) of siRNA, and mix gently.
2. Dilute Transfection Reagent: In another sterile tube, add 125 μL serum-free medium and 5 μL Hieff Trans™ Booster Transfection Reagent. Mix gently.
3. Combine and Incubate: Add the diluted siNA solution to the diluted transfection reagent tube. Mix gently by pipetting. Incubate at room temperature for 10–15 minutes to form siNA-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
SiRNA was transfected into primary glioblastoma cells(GBMs) using Yeasen Booster and a commercial L*2000 transfection reagent. Knockdown efficiency was assessed by Western blot (WB) analysis.
Figure 1. SiRNA transfection inprimary glioblastoma cells(GBMs) using Booster DNA&RNA Transfection Reagent versus L*2000. The result show that Yeasen Booster transfection reagent achieves efficient transfection and 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 |
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