siRNA againts Beta Actin Help PLease - (Mar/10/2010 )
I'm currently working on regulating Beta Actin using a plasmid by ambion called pSilencer 2.1 U6 Hygro. I got the plasmid to cut open using BAMHI and HINDIII sites but i now need to create a siRNA oligo. THe most common method for siRNA seems to be lipofection or electroporation. However, my professor is aiding me in lipofecting this plasmid in, and then creating selection pressure using hygromycin to select for the plasmid. Does lipofecting or electroporating sound like a good idea? as lipofecting/electroporating the siRNA oligo seems to be the most popular method. Please give me input!
I will confirming the data by doing qRT-PCR. Thank you
It depends on the purpose of your experiments, and the type of cells you work with. siRNA mediated RNAi is transient, but every easy to do, costs similar to shRNA vector. If you want to have long-lasting RNAi effect, or your cells are hard to transfect, nondividing etc, then constructing your shRNA vector which may be very time consuming.
we've transfected our HeLa cells with gfp and a bunch of plasmids easily before. yes, this will be transient, and i am thinking about making a stable transfection by titering hygromycin(plasmid has hygro resistance) and having it integrate. this will be cheap and easy, since i got a crelox plasmid that might take a while to come in. Thank you pcr man! btw, this actually might be a shRNA vector. my siRNA construct looks like this:
see my flikr site: http://www.flickr.com/photos/48416970@N04/...57623499021073/