I have generated and validated a mammalian expression vector-based amiRNA library. The validation was performed in over-expression experiments in cell lines, cotransfecting the amiRNA vectors and the target fused to Luciferase, this to detect the Luc signal in cell lysates. In these tests 24-48 hours is enough to get a strong knock-down effect. However, I will now screen my library in primary neurons to look for specific phenotypes. Could anybody help me and give me some advices regarding how long I should express my constructs to get or at least to assume that I could get a decent knock down?
Thanks in advance!
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amiRNA expression time required in primary cultures.
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