I work with siRNA transfection in endometrial cancer cells where 72 hours after siRNA transfection, the cells reach a maximum of 65% to 70% confluency. In contrast, 95% to 100% confluency is required in a scratch assay. Please let me know what protocol this assay follows after siRNA transfection. Is SiRNA transfection performed before scratch making or after making of scratch? (because transfection is ideally performed for 50% to 60% of cells?)
Hello Joydeep. Ideally, si RNA confirms the transient transfection which lasts for 48-72 hours. Confirming the KD and setting up the process may take time. Instead, you can use the shRNA system/lenti viral and select the transfected ones. For seeding the cells for scratch assay, 6 well plate is ideal, and seeding more cells to get 85-90% confluency the next day before scratch.
You can go through the attached article.
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