Why there is no knockdown? - (Feb/19/2010 )
1.I have this shRNA sequence contained in PLKO.1 vector. I transfected the shRNA into suspension neurosphere cells using fugene 6. 6hrs later, media was changed to fresh media. 48 hrs post-transfection, media was replaced with media containing puromycin (used concentration obtained from puromycin kill curve). Left in incubator untouched for 6 days. 6 days later, most cells look dead. Proceeded to do FACS sorting to sort out the live cells using the scatter gate. Extracted RNA using Cells Direct Kit from Invitrogen and performed RT-PCR using taqman probes. However, there was no knockdown detected. The shRNA used is validated to give a knockdown by the company.
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)?
It sounds like your transfection efficiency is very low. Do you have any GFP marker that you can observe, or a positive control like gapdh?
Dont assume the knockdown as it is validated by a company. If there is knockdown of the mRNA, you should be able to pick it up by QPCR.
Try other shRNA sequences also as controls or simply to comapre knockdown efficiencies.
Your plasmid DNA has probably diluted out during the time course of selection. pLKO does not have a eukaryotic origin of replication so every time your cells divided your plasmid was halved between the two daughter cells. Also plasmid levels drop over time as they are degraded by the cell.
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...