Reagents List
|
Product Name |
Cat.NO. |
|
Hieff Trans™ RNAiBoost Transfection Reagent (RNAiMax alternative) |
40807ES |
THP-1 siRNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Cell Counting: Remove cells from the incubator and mix the cell suspension thoroughly by pipetting up and down. Use a cell counter to determine cell viability and density.
- Cell Subculturing (Direct Dilution Method): Based on the counting results, passage the cells by diluting them to a density of approximately 5 × 10^5 cells/mL.
- Cell Subculturing (Half-Medium Exchange Method): If the supernatant of the culture appears yellowish, allow the cells to settle undisturbed after removing the plate from the incubator. Tilt the plate slightly to let the cells settle at the bottom, then aspirate the supernatant using a pipette. Finally, add fresh medium, resuspend the cells, and proceed with counting and passaging.
- Cell Subculturing (Centrifugation Method): If the cell condition is poor, passaging via low-speed centrifugation is recommended. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 200 ×g for 3–5 minutes. Resuspend the pellet in fresh medium, then count and passage the cells.
Cell Transfection (12-Well Plate)
I. Preparation
1. Cell Preparation: 24 hours before transfection, digest cells with trypsin, resuspend, and perform cell counting. Seed cells in 12-well plates at a density of 5E5 cells/well in 1 mL of complete medium. The target confluency at the time of transfection is 70%.
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.
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 THP-1 human monocytic cells and induced M0 macrophages was performed using Yeasen RNAiBoost, followed by qPCR analysis to evaluate transfection efficiency.
Figure 1. siRNA transfection in THP-1-derived M0 macrophages was performed using Yeasen RNAiBoost. The results showed that the knockdown efficiency in these induced M0 cells exceeded 80%. This demonstrates that Yeasen RNAiBoost facilitates highly efficient transfection, 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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