Dear all,
I am working on a microRNA subject. The microRNA could inhibit cell proliferation, and then i detected the colony formation ability. Compared to negative control, cells transfected with my target microRNA formed less colony than negative control for the first several days,but after 12 days, some colony became very large while some cells still could not form colony. The colony size of the negative control group is relatively uniform. Anyone knows what's the problem? Or if it is possible that the cells transfected cells secrete some molecules stimulate the colony formation of untransfected cells?
Thank you!
Some more info would be helpful jenny. I assume that you are transfecting both groups of cells, so all of them are transfected? the negative control in this case should be a control scrambled miRNA. If that's what you are doing, my guess is that your problem is the transient nature of your transfection. colony formation assays would work much better if you do this using a viral vector.
I agree with Panos's response. If you have a stable expression system, you may not have this issue.
I would avoid generating stable polyclonal populations. this could allow the cells to circumvent whatever pathway you are inhibiting. Infect fresh cells each time.
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