protein did not knock down by shRNA, but RNA did knock down
Posted 29 September 2011 - 11:49 PM
our lab encounter a problem in the experiments. when we used two independent shRNA to knock down the target genes, the RNA knockdown well (about 70-90% knockdown), the phenotype of the shRNA which promote cell differentiation or block cell selfrenewal is also changed very dramatic. In addition we also observed the changes of the downstream pathway. however, the protein level of the target gene did not change. we harvest the cell after 4,6,9,15 days post transduction all shown similar results. could somebody help us to resolve this issue? is the protein halflife problem or the shRNA problem? why we can observe the same phenotype with at least two shRNA if it is off-target effect?
thanks for your kind help!!
Posted 30 September 2011 - 06:31 AM
Gene Tools, LLC
Posted 01 October 2011 - 01:37 PM
Posted 02 October 2011 - 08:15 PM
thanks a lot! one of my protein is the transciptional factor GATA4 , the other is apolipoprotein 1. we did not know about the half life of the protein. we are sure the RNA still knowdown till day 9. the shRNA primer should be fine. we tried three antibodies fir the GATA4 and had the same results. we are really puzzle about these.
please do not hesitate to let me know your opinions. we appreciate your kind help!
Posted 08 October 2011 - 10:42 PM