transient transfection problem using ME180 cells - (Oct/17/2012 )

Hi,

I've generated a few shRNA expression vectors to knockdown uPAR. uPAR is upregulated under hypoxia conditions, and I am trying to verify that my knockdown vectors are indeed inhibiting expression of uPAR in ME180 cells under hypoxic conditions. Therefore, I am performing transient transfection reactions, extracting RNA from the cells after treatment under air vs hypoxia (using controls), and then doing RT-PCR to determine levels of mRNA for housekeeping genes (L32 and YWAZ) and uPAR. However, I keep running into the same problem, in that there is no upregulation of uPAR in my hypoxia cells, and there is also no decrease in expression of any of my shRNA vector transfected cells. The results show a consistent level of uPAR mRNA in all of my cells regardless of what condition I put them through. (The Ct values are consistently all around 17-19). Could you please give me some suggestions as to what I can try and do to circumvent the problem?

Thanks!

Perhaps the induction of hypoxia isn't working. Try a different method.

Perhaps uPAR is already upregulated because of something you are doing during the culture (heat/cold shock?), or because it has a role in the proliferation of the cells?