Hi,
I have an question regards soft-agar colony-forming assay, if firstly transit transfected miRNA mimic, then start soft-agar colony-forming assay, does that works? this looks wield for me, since miRNA mimic usually function around 72h while soft-agar colony-forming assay usually need 2 weeks?
I thought it is irrational, but i do find some paper did this, does some has experience on this, or frist siRNA transit transfection, then soft-agar colony-forming assay? thank.
or simplified question, does soft-agar colony-forming assay must done in stable cell lines, or could also in transit transfected cells? Thanks.
Best,
Ken
Like drug treatment every 72 hours you can replace your old media with miRNA mimic by new, by this way you can get rational output.
I also recommend you to use materiel instead of soft agar for colony assay.
Thanks Lqbal, Your idea is good, i also think the soft agar is not suitable for this purpose, Thanks. And if i really need to do soft agar, probably stable cells is better choice.
Best.,
ken
Hi Kecheng,
I would try to generate a stable cell line, or an inducible stable cell line, if possible. Once the colonies are formed, I am concerned of the efficiency of the siRNA or the miRNA mimic reaching all the cells in the colony.
Another point is setting up the soft agar experiment once the miRNA mimic/shRNA is having an effect (you pointed to 72h) because otherwise the effect of your miRNA/shRNA on the ability to form colonies will be mask by incomplete effect of the miRNA/shRNA on the cells.
I hope this helps,
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