hi guys, I tried to transfect lentiviral based shRNAs into 293T cells and subsquently infect into stem cells. My transfection and infection looks 70% and 90% positive respectively based on GFP expression. But still don't get my knockdown (from WB). I had tried two shRNAs few weeks back and one of them had worked but when I tried other constructs including the one that worked for the first time as a positive control, none of them produce knockdown. have you ever had similar experiences? is there any possible way to troubleshoot ? does changing target cell help?
Another couple possibilities:
1) The band your antibody shows you may not be the protein you think it is
2) The shRNA may simply not be an effective sequence (try another one)
3) The shRNA might not target the primary mRNA variant in your cells
Of course you could rule some of these out by performing RT-qPCR to see how efficiently you are knocking down the mRNA you are targeting
You have a random integration of the shRNA into each cells. Even if all stem cells get infected you might not have a knockdown since the shRNA is not generated in all the cells. I had this problem too. Thus, I started with clonal expansion and everything worked well. If you need a protocol for clonal expansion let me know. Otherwise you will find good ones in the literature.
Thanks guys, I know I am responding more than an year late, but all of your recommendations helped me a lot. Thank you.
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