| Instructions For Use |
The following protocol uses HeLa cells as an example and assumes a DMEM-based culture medium.
1. Seed 5 × 10^5 HeLa cells in a 35 mm tissue culture dish containing 2 mL growth medium (DMEM + 10% FBS + 1% penicillin/streptomycin). Cells should reach 50–60% confluence the next day.
2. 24 hours after seeding, pre-warm the shRNA lentiviral medium to 37 ℃.
3. Remove 0.5 mL of the original medium from the dish.
4. Gently mix the lentiviral solution three times using a serological pipette.
5. Carefully add 0.2 mL of lentiviral solution to the dish, dispensing along the wall to avoid splashing.
6. Add polybrene to a final concentration of 5 µg/mL and gently swirl to mix.
7. 48 hours after seeding, add another 0.2 mL of lentiviral medium directly to the dish without removing the existing medium.
8. Add additional polybrene to maintain a final concentration of 5 µg/mL.
9. 72 hours after seeding, collect the cells by aspirating the medium, rinsing once with PBS, trypsinizing, and transferring the cells to a 60 mm dish.
10. Add puromycin to a final concentration of 4 µg/mL for selection. Include a dish of wild-type HeLa cells as a negative control to monitor puromycin effectiveness.
11. Continue puromycin selection for 48 hours. Most wild-type cells should die, while infected cells should survive.
12. Replace with fresh growth medium containing 2 µg/mL puromycin and culture until cells reach confluence before harvesting or staining. If cells are highly sensitive to puromycin, regular growth medium may be used at this stage.
13. For RT-qPCR, collect 1 × 10^4 cells and extract total RNA. At least 48 hours after the medium change in Step 12 is required to allow sufficient time for mRNA degradation.
14. For protein analysis, collect 1 × 10^6 cells in lysis buffer and load 30 µg total protein from both wild-type and knockdown samples to evaluate target protein knockdown by Western blot. At least 5 days after the medium change in Step 12 is required to allow sufficient time for protein degradation.
Tips:
1. For genes and proteins with long half-lives, to ensure sufficient knockdown, the sampling time in Step 13 may be extended to 72 hours for mRNA analysis, and the sampling time in Step 14 may be extended to 7–10 days for protein analysis.
2. Optimize polybrene concentration and puromycin dose for other cell lines before routine application.
3. Handle lentiviral materials using applicable biosafety procedures and institutional requirements.
4. Because transduction efficiency varies among cell types, the optimal MOI should be empirically determined for each specific cell type or cell line. We generally do not recommend a single MOI for any given cell type. Instead, upon receipt of the product, we suggest testing a range of lentiviral volumes, such as 200, 100, 50, 10, and 5 µL, to transduce the cells. After transduction, apply puromycin selection to enrich for cells with shRNA integrated into the genome, and then assess knockdown efficiency by qPCR or Western blot. Finally, choose the lowest viral dose, corresponding to the lowest effective MOI, that provides acceptable knockdown while maintaining good cell viability. |
