Cannot get good knockdown with multiple shRNA constructs - (Aug/24/2012 )
I've been trying to knockdown my protein of interest using some shRNA constructs from Origene (HuSH 29-based). I received 4 different constructs, but no single construct is able to give me more than, at most, a 30% knockdown. While I'm seeing some changes in cell viability in my knockdowns vs. scramble, I'm really hoping to get a much stronger change with a stronger knockdown. Since at least two of my constructs cause a 30% decrease, would it be okay to co-transduct viral particles containing the two constructs? I'm concerned that mixing the two viruses together is going to add confusion and complexity, but I would like to get a stronger knockdown. Would that help, or is there something else I can do to increase knockdown?
I believe the shRNAs you purchased from Origene have been validated to have good knockdown efficiency. Have you checked your infection efficiency and the quality of your packaged virus?
It is fine that you combine several shRNA targeting the same genes together to get better knockdown. A potential drawback is that if they each have some off-target effect, combining them will result in greater off-target effect. Ideally, all shRNAs should give you decent knockdown, while giving you similar functional consequences.
I do have same problem, but while picking up the colonies, I guess I have heterogeneous population of cells ( both wild type cells and knockdown cells in the same population). I am sub-culturing them, hoping that my knockdown levels will go down. I have around 74% knockdown for few of the clones at this time. I will update once I get my real time data done.
I had the same problem with one of my cell cultures, so I sub-cultured them and I managed to get a better knockdown efficiency, 60% to 85% ! Hope it will work for you too.
i would look at the protein Kd levels, sometimes 30%down on rna might yeild significant translational down reg.