mRNA induction after shRNA transfection - (Jul/08/2007 )

Hi.

I am trying to knockdown a gene in chick cell lines. A different problem I have is that even after trying a lot of reagents, the best transfection I have gotten is about 30-35%.
I ran with this regardless, and tried 4 shRNA constructs. I got one to work better than the others, and after transfection, I have been getting a consistent ~30% knockdown at 24hrs as compared to my control LacZ-shRNA.
So, I am guessing that its safe to assume that the construct is knocking down my gene of interest in the cells that it gets into.

However what I found peculiar was that in both the gene directed as well as the control shRNA transfected samples, there is between 1-3 fold increase (again, that is consistent in the 3-4 times that I have repeated the expt so far) as compared to the non-transfected control. When I transfected with a non-shRNA, GFP containing plasmid, I found a similar, but milder induction in gene expression. I am using b-actin as an endogenous control to normalize my fold expression values.

I was wondering if anyone else has seen something like this. Could this be a gene specific effect, i.e. maybe the gene is induced following stress to the cells?? or is there something else known about this!!

Also, when I collect cells at 48hrs, the values are back to normal... just like the non-transfected control!! I have read that shRNA's should repress expression for 4-5 days, right. I am guess its just an effect of the cells growing/dividing, and so the initial 30% is then reduced to a non-significant number of cells with the plasmid!?! I am going to try and select next, and I am in the process of making viruses as well,,.... but I was wondering if anyone had any idea or any other explanations about these issues.

Thanks in advance for any helpful suggestions,

Vik.

There is one published observation for this that I am aware of; see Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17337-42. Epub 2006 Nov 3. PMID: 17085592

The fact that you don't see continued activation for longer than 48 hours is strange though, since the above paper demonstrated a long lasting activation. maybe it is a non-specific cause in your case?

Also, I'm confused about why you see a decrease in one experiment and increase in another can you explain the experiments you performed? ("...and after transfection, I have been getting a consistent ~30% knockdown at 24hrs as compared to my control LacZ-shRNA." and then you say "...there is between 1-3 fold increase (again, that is consistent in the 3-4 times that I have repeated the expt so far) as compared to the non-transfected control").

Thanks miRNA man.

Sorry, I know what I said was a little confusing.

I have 2 controls, a shRNA control (LacZ-shRNA), and a transfection control (non-transfected samples).

So, when I compare the mRNA expression level in samples transfected with my target sh-RNA construct with the control LacZ transfected samples, I see a 30% reduction, consistent with the %of cells that I can get the plasmid into. So, I am guessing that my construct is knocking down the gene in the cells that its getting into.

But the levels of the mRNA of my gene of interest are 1-3 fold higher in BOTH my test construct, and the control LacZ construct transfected samples.

I am not sure if I am very clear even now.

It makes it clearer, thanks. It sounds like some non-specific or transfection-dependent things going on. Do you see the same effect with siRNA with the same sequence, instead of shRNA? Perhaps you could think about an inducible system (i.e. pSICO or a tet-ON/OFF system), then you can transfect, wait for the effects of transfection disapear and at the same time select for stably transfected cells, although that might just be too much effort. Cloning takes me a long time compared to most on this site.