Reagents List
|
Product Name |
Cat.NO. |
|
Hieff Trans™ RNAiBoost Transfection Reagent (RNAiMax alternative) |
40807ES |
HK-2 siRNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Washing: Aspirate the old culture medium, add 3–4 mL of room-temperature PBS, gently shake 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 cell gaps widen and cells become rounded, gently pipette with trypsin to dislodge the cells.
- Termination: Add 2–3 mL of complete culture medium to stop digestion, then pipette to mix and form a uniform cell suspension.
- Seeding: Transfer the suspension to a centrifuge tube, centrifuge at 200 ×g for 3–5 minutes, discard the supernatant, resuspend the cell pellet in fresh culture medium, seed into new culture flasks, add appropriate volume of medium, and continue incubation.
Cell Transfection (12-Well Plate)
I. Preparation
1. Cell Preparation: 24 hours before transfection, cells were trypsinized, resuspended, and counted. Cells were seeded into 12-well plates at an appropriate density (5 × 10^5 cells/mL) with 1 mL of complete medium per well. The goal is typically to achieve 60% - 80% confluency at the time of transfection.
Cell density during transfection in this experiment: 40–50%.
2. Reagent Preparation:
- siRNA: Synthesize high-quality siRNAs. Typically, three distinct siRNAs are designed for validation, with the initial concentration adjusted to 20 μM.
- Transfection Reagent: Allow Hieff Trans™ RNAiBoost 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. Cell Transfection Protocol
1. Dilute siRNA: Pipette 2.5 μL of siRNA solution and mix thoroughly with 12.5 μL of Enhancer solution by pipetting up and down.
2. Dilute Transfection Reagent: Add 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 30 μL of Opti-MEM transfection diluent to the mixture from step (2) and mix well. Add the resulting 50 μL transfection complex to one well of a 12-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 1 mL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional.
2. Analysis: Typically, RNA is extracted for analysis 48 hours after transfection. In this experiment, qPCR was performed 48 hours post-transfection. In this experiment, qPCR analysis was performed 48 hours after 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
siRNA transfection in HK-2 Human renal proximal tubular epithelial cell was performed using Yeasen RNAiBoost, and transfection efficiency was evaluated by qPCR at 48 hours post-transfection.
Figure 1. Comparison of transfection efficiency and cytotoxicity between Yeasen RNAiBoost and Lipofectamine 2000 in HK-2 cells.
HK-2 human renal proximal tubule epithelial cells were transfected with siRNA using either Yeasen RNAiBoost or L* 2000. Cells treated with RNAiBoost exhibited better cell health with significantly fewer vacuoles compared to the L* 2000 group. In terms of knockdown efficiency, all three siRNAs delivered by RNAiBoost achieved approximately 90% silencing, whereas the highest efficiency observed with L* 2000 was only around 70%. These results indicate that Yeasen RNAiBoost facilitates highly efficient transfection with lower cytotoxicity, leading to a significant reduction in target gene expression.
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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