Why there is no knockdown?
Posted 19 February 2010 - 06:06 AM
2. Can taqman probes in real-time PCR be used to detect for knockdown? Do the primers need to cover the region cleaved by the RISC complex?
3. Any other suggestions about how to do RT-PCR for a small number of cells (about 200 cells)?
Posted 19 February 2010 - 08:58 AM
Posted 19 February 2010 - 11:55 AM
Try other shRNA sequences also as controls or simply to comapre knockdown efficiencies.
Posted 25 February 2010 - 06:57 PM
It also looks like your efficiency was low which usually means that your cells did not take up DNA efficiently.
Why not package the vectors into viruses? Lentiviruses are fantastic for difficult to transfect cells and you will get stable integration in the genome so you won't lose expression of shRNA every time your cells divide.
1. Taqman is just fine for detecting knockdown. The entire mRNA sequence will be targeted for degradation so it really doesn't matter where your primers are.
2. Yuck, amplify your RNA with a kit???? Not sure on this one...