protein did not knock down by shRNA, but RNA did knock down - (Sep/30/2011 )
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!!
I wonder about the stability/turnover rate of the protein and the duration of good knockdown. You checked protein levels at 4, 6, 9 and 15 days post-treatment but did you also check the mRNA to see whether the knockdown is being maintained that long? What did you find? What do you know about the protein turnover --- is this a transcription factor with rapid turnover or a structural protein which is very stable?
I would also be concerned about the specificity of both your antibodies and your shRNA.
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!
If the protein isn't down but you are absolutely sure that the RNA is down, then there are three options: 1) your antibody isn't picking up what you think it is. 2) Your shRNA isn't really knocking down the RNA - it is targetting something else (what about pseudogenes?). 3) only low levels of the protein are being produced at any one time, so it only needs a low level of RNA.