I am currently going through my options for knockdown and overexpression of genes in cell culture. I am interested in the shRNA option but have some questions as have no experience of using this method.
Is there a commercial company from which you can buy ready to use (ie add to your cells) shRNA?
You can get shRNA as packaged lentiviral particles from Sigma, Santa Cruz, and many others - just put them on your cells and do analysis a few days after transduction. If your cell line tolerates transfection well, just get the shRNA plasmids and transfect them in as that is much more cost-effective. You may want to try using a comparison-shopping webisite like Biocompare, or ask a group that has successfully knocked down your gene of interest to see what method they used. Good luck!
I have good and bad experiences with Sigma Mission shRNA products (http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/shrna-search-and-order.html), they are quite simple to use, for example you can buy either the plasmids, the already transformed bacteria in a glycerol stock or the lentiviral particles (probably what you want). You can also buy them as a pack of five plasmids with different sh targeting sequences. However, although I found one protein very easy to knockout using this system, a second different protein proved very difficult.
shRNAs come in different types of vectors. I tried many types and got the best results with the pLKO.1 backbone also called TRC (TRC1 or TRC2). This type of shRNA vector is sold by Sigma and Open Biosystems. Usually if you order more than 5 you get a good discount. Also, if you can afford it, order more than one control shRNA since even if they should not have any effect on your cells, some of them still do. If you are not in a rush, I would not get the transduction-ready viral particles because not only they are more expensive but more importantly you may need to optimize the amount of viral particles you apply to your cells and also the incubation time. And for this optimization step, you may not have enough with what they send you. For example, too many viral particles incubated with your cells may kill your cells. Too little may not be enough to infect all your cells. To optimize the conditions of your knock-downs, it is better to prepare your own viral particles with the vectors containing the sRNAs these different companies provide. Getting the bacterial glycerol stock is usually cheaper. Then you prepare DNA from these bacteria, transfect HEK 293T cells (with the packaging vectors as well) and infect your cells with the 293T medium (different amounts, times...). If you need a good protocol, let me know.
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