Lately I have been transfecting human embryonic stem cells (hESCs) with an LNA against a specific miRNA (let's call it miR-X). As a control, I use both cells that are transfected with a control LNA from exiqon (ctA-LNA) but also non-transfected (let's call them NT) cells. The first time I carried out the transfection (with lipofectamine2000), it seemed, (both in terms of mRNA expression and miRNA expression profiles), that my ctA-LNA-transfected cells were very much close to my NT cells.
When I went on to repeat the experiment however, I started seeing major differences. When transfecting with the miR-X-LNA, the elimination of miR-X was indeed almost complete when compared to ctA-L-transfected cells, but the expression levels of miR-X, as well as the expression of several other mRNAs I tested were also downregulated in ctA-L cells when compared to NT cells! And in some cases the downregulation was pretty big (~50-60%)!
I stopped thinking that there is something wrong with my hands since I repeated this many times, and saw the same thing. I am now more prone to believe that in general, transfection with a small RNA molecule, disturbs cells overall, and that there is simply nothing we can do about it.
However, I still wonder which comparison is more meaningful? Should one compare the miR-X-transfected cells with the ctA-L-transfected or the NT cells? I have discussed this with a colleague who believes that the comparison to ctA-L is more "appropriate" since we have to merely "accept" that transfection with such molecules "by default" has some sort of effect on the cellular system and as such, this should be the starting point for any comparisons.
Your last paragraph says it all Marilena. You got to accept whatever effect you get with your control compared to the non-transfected cells. By no means you can consider "untransfected" cells as your control, because transfection reagent as such exert some effect on the cells, leave aside the control miRNA or anything else.
You can either ask exiqon if they have some alternative control LNA or you will have to try your experiment with some other miRNA expression system apart from Exiqon to get rid of the undesired effect of control miRNA transfection.
I found this type of effects in several occasions. I agree with Ashraf that the Non-transfected condition can only be the control for your LNA-control, I mean, that if your LNA-control doesn't behave like your non-transfected cells, then is not a good control. I usually perform the experiments with two non-silencing controls. In particular cell types, both of them behave as untransfected cells, but sometimes, one of them may have an effect. I must say that this type of effects have been also shown with miRNA mimmics controls of Dharmacon (now Thermo), where they do not hide that one of their controls may target EGFR receptor.
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