I used an X-treme siRNA transfection reagent to transfect miRNA. After that I performed Real-Time PCR to validate my transfection. I do my transfection at 2 different times (8 hour and 72 hour). Normally, miRNA level is higher in 8 hour than 72 hour. However, in my Real-Time PCR miRNA level is higher at 72 hour than 8 hour. What is the problem?
Darrell is right, it depends on the miRNA, as well as on the system you use to over-express it, whether it is a vector expressing the pre-miRNA or a miRNA mimic. I normally use synthetic miRNA mimics, and the first thing I always do is a dose dependent time-course assessment, so transfection of different amounts of the mimic and collection at different time points (typically 24, 48 & 72 hours).
I would say 8 hours is a bit early, which system did you use to over-express your miRNA?
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