Reagents List
|
Product Name |
Cat.NO. |
|
Hieff Trans™ RNAiBoost Transfection Reagent (RNAiMax alternative) |
40807ES |
HEK293 saRNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Washing: Aspirate the old culture medium. Add 3–4 mL of room temperature PBS, gently swirl to rinse for 10–20 seconds, and then aspirate.
- Digestion: Add 1 mL of trypsin to wet the bottom of the flask and incubate at 37°C for digestion.
- Detachment: When the intercellular spaces widen and the cells become rounded, gently dislodge the cells by pipetting directly with the trypsin.
- Termination: Add 2–3 mL of complete medium to terminate the digestion. Pipette gently to mix and form a uniform cell suspension.
- Seeding: Collect the suspension and centrifuge at 200 ×g for 3–5 minutes. Discard the supernatant, resuspend the cell pellet in fresh medium, subculture into new flasks, top up with medium, and continue culturing.
Cell Transfection (24-Well Plate)
I. Preparation
1. Cell Preparation: 24 hours before transfection, digest cells with trypsin, resuspend, and perform cell counting. Seed cells in 24-well plates at a density of 5E5 cells/well in 500 μL of complete medium. The target confluency at the time of transfection is 60-80%. In this experiment, transfection was initiated when the cell confluence reached 50%–60%.
2. Reagent Preparation:
- saRNA: Prepare the saRNA required for the experiment.
- Transfection Reagent: Remove the RNAiBoost reagent from the 4°C refrigerator and allow it to warm up to room temperature. Gently pipette or flick the tube to mix before use.
- Serum-free Medium: Prepare the basal medium (e.g., Opti-MEM) that will be used to dilute both the saRNA and the transfection reagent.
II. Cell Transfection Protocol
1. Dilute saRNA: Pipette 1.25 μL of saRNA solution and mix thoroughly with 6.25 μL of Enhancer solution by pipetting up and down.
2. Dilute Transfection Reagent: Add 2.5 μL of RNAiBoost in vitro transfection reagent to the mixture from step (1). Mix thoroughly by pipetting and incubate at room temperature for 5 minutes.
3. Cell Transfection: After incubation, add 15 μL of Opti-MEM transfection diluent to the mixture from step (2) and mix well. Add the resulting 25 μL transfection complex to one well of a 24-well plate. Gently rock the plate to ensure even distribution. The complex can be scaled up according to the number of wells required.
4. Cell Culture and Detection: Return the cells to a 37°C, 5% CO₂ incubator. Assess the gene silencing efficiency 24–48 hours post-transfection.
III. 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 500 μL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional. The culture medium was replaced 6 hours post-transfection in this experiment.
2. Analysis: Typically, RNA is extracted for analysis 48 hours after transfection. In this experiment, fluorescence imaging was performed 20 hours post-transfection.
Tips:
1. If significant cytotoxicity is observed after transfection, consider replacing the medium at 6 hours or halving the reagent dosage to avoid cytotoxicity issues.
2. If the transfection efficiency is relatively low, increasing the amount of transfection reagent can effectively improve it.
3. Opti-MEM is recommended for diluting siRNA and transfection reagents.
4. The transfection reagent is compatible with serum and antibiotics; however, the medium used to dilute the plasmid and transfection reagent must be serum-free and antibiotic-free.
5. Strictly adhere to the recommended dosages in the table for preparing the transfection complex. If you need to increase or decrease the dosage, do so proportionally. For example, for a 6-well plate, if the final concentration is 50 nM, prepare 100 µL of transfection complex according to the manual. If you need to double the dosage, prepare 200 µL of transfection complex and add it to the well. Conversely, if you halve the dosage, simply add 50 µL of the prepared 100 µL transfection complex to the well.
Experimental Results Analysis
In this experiment, HEK293T human embryonic kidney cells were transfected with saRNA using the Yeasen RNAiBoost Transfection Reagent, and the transfection efficiency was evaluated via fluorescence microscopy.
Figure 1. saRNA transfection in HEK293 cells was performed using Yeasen RNAiBoost. The results demonstrated that the Yeasen RNAiBoost reagent achieved highly efficient transfection in HEK293T cells.
Guidelines for RNAiBoost Reagent Dosage in Different Culture Dishes(for reference only):
|
Culture Plate |
Culture Medium Volume |
siRNA Solution (Stock 20 μM) |
Enhancer |
Transfection Reagent |
Serum-free Medium Volume |
Transfection Complex Volume |
|
24-well plate |
500 μL |
1.25 μL |
6.25 μL |
2.5 μL |
15 μL |
25 μL |
|
12-well plate |
1.0 mL |
2.5 μL |
12.5 μL |
5 μL |
30 μL |
50 μL |
|
6-well plate |
2.0 mL |
5 μL |
25 μL |
10 μL |
60 μL |
100 μL |
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