Hi,
I silenced a gene (LSD1, KDM1A) using 2 different shRNA in 3 different cell lines. When I check its mRNA level, it is more than 70-80% silenced. However, if i check its downstream target (at mRNA level), they are not affected, unlike papers in literature. According to the papers, they should be up or down-regulated. I checked 7 target genes. All are unaffected, only itself(LSD1) is downregulated (70-80%). Do you have any similar experience or suggestion about what may cause this problem? Is it technical or biological (specific to cells I tested)?
Thanks,
Burak
Have you checked your knock down at the protein level?
The mRNA might be down but not the protein: the KDM1A protein might have a long half life.
If you want to see downstream effects you need the protein to be depleted. You should analyze your depleted cells by western blot and check whether KDM1 is also down.
Moreover, sometimes the 20-30% leftover (protein) after depletion can be enough to carry out the function.
with good, potent siRNAs and optimized delivery one can easily accomplish >80% knockdown at the mRNA level. but at the protein level its usually much less. So you might further boost knockdown by using either higher siRNA concentration, or find a better design. However, keep in mind- when you are trying to replicate someones data, not necessarily this will happen. They might be using some unique system which is not trivial to replicate, or it could have been one quick and dirty experiment, published right away, and the results are not real...
Hi
You should check your knock down at Protein levels too.
Then only you can see the effect of your experiment.
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