Best way to detect shRNA produced in cells? - (Jul/08/2006 )
can anyone suggest a method for measurement of the level of shRNA made in stable-transfected cells? Thanks.
Since transcribed shRNA is supposed to be cleaved to form siRNAs, it likely would not be possible to directly measure your shRNA of interest via PCR/northern. You can get a general feel for level of expression by generating a stable line with either the same promoter and GFP or anti-lamin shRNA and measuring those instead. Depending on integration site, however, there may be considerable deviation in the expression levels.
If its important to know and control the amount of shRNA expressed in your cells, I would alternatively suggest looking into a tet-inducible shRNA vector. In this case you can relate shRNA expression level to the amount of tet added to culture media.
Edit: one more possibility is to use a dual-expression shRNA vector. Use one side for your shRNA and the other for a target you can measure for expression level.
Thanks. Has anyone tested shRNA using mirVana™ from Ambion? I'd love to know how does it perform.