We recently used siRNA to knock down a gene in the HCT116 cell line. The knockdown had no effect on target gene expression, whereas overexpression had a minor effect in the same cell line and reduced target gene expression. Any explanation or mechanism might be used to explain such a situation.
"your knockdown didn't work, and your overexpression was toxic"?
I mean, there's a lot of detail needed here that you're not providing, but sometimes the simple answers are the best.
How do you know your siRNA K/D worked? The evidence suggests that it just...didn't. They don't always work, so this is worth confirming.
How do you know your overexpression wasn't toxic? If lots of your cells are dead, expression of your GOI will go down overall.
Your question seems incomplete. Still, as you mentioned that overexpression worked and "affected" the target gene, I am supposing that your gene of interest whom you are doing overexpression/ KD is different and the other one is its target.
So in that case, there are several mechanisms to explain.
For example, several genes stimulate their own expression as positive feedback. such as transcription factor NRF2 regulates itself too.
There can be an opposite case too.
Further, a conventional method doing such studies is that you first compare the gene (which you are doing KD) in different cell lines, and pick the one/two which has its "comparatively" lower expression for overexpression (opposite for KD).
In end, there is still a lot of assumptions on which we have to work on. It will be better if you provide more detail.
And yes...that Knockdown didn't work.
John Hildyard Thanks for the answer,
Actually, we just used qPCR to detect knockdown or overexpression efficiency but we didn't check for the toxicity.
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