is there any fast screening after siRNA knockdown of protein? - (Sep/14/2006 )
I am doing a siRNA knockdown of a protein in HepG2 cells.
after transient transfection, western blotting was done and shown that the desired protein amount was reduced.
however, after selection with g418, the knockdown effect was failed, also, cells appeared to be resistant to G418.
i would like to ask, as i hav read some picking clones technique in this website (i found it's pretty useful!!), after using trypsinized paper or dilution methods to make "monoclone", with such a small no. of cells, (suppose i did it in 96 well plate) how can i screen the cells if the protein is still knocked down or not? and moreover, it should be done after one week as it seems showing drug resistant to G418 when time went.
really thanks for the help!!
Are there any morphologic changes after the protein knockdown? If yes, that can be indicative of positive clones.
I know that some realtime RT-PCR systems allow expression analysis of a single cell.
Hi, please correct me if i'm wrong. you mentioned siRNA, these come as duplexes and are normally not cloned into vectors. Why had you perform selection with G418?
However, if you had cloned into a vector that's neomycin resistant, you should be picking a couple of clones. From your 96plates, transfer them to petri dishes and grow them to a large enough density for western blot, etc. I'm not sure of any fast method!!
In regards to what was mentioned above, you could do a dot blot to speed things up a bit.