miRNA knockdown experiment controls - Very confused (Jun/16/2011 )

Hi all,

Just started down the track of miRNA knockdown experiments (injection of miRNA into embryos) in zebrafish and I'm a tad confused. I've included the obvious controls: uninjected, eGFP miRNA injection (WT fish has no eGFP expression) and scrambled miRNA injection. I've done real-time PCR to assess for knockdowns and I'm getting really "interesting" results. There is very little knockdown of mRNA for my gene of interest when I inject different concentrations of miRNA (compared this with uninjected control), but assessment of the eGFP/scrambled miRNA injected embryos showed that the expression of my gene of interest in these samples were much lower than the uninjected controls. Does this mean that the latter miRNAs are knockdown down my gene? Should I be comparing my results with the uninjected control or eGFP/scrambled injected embryos? I've checked the literature and people seem to do both of these things.

In principle, eGFP/scrambled miRNA should be used as controls and compared to. But you really need to see some downregulation compared to uninjected controls to be confident that it is working.

pcrman on Mon Jun 20 04:24:12 2011 said:

But if your eGFP/scrambled miRNA control is causing your gene of interest to increase or decrease is that not concerning? There is some downregulation (very little) of my gene of interest when compared to uninjected control, but when that same sample is compared to the eGFP/scrambled the expression is of eGFP/scrambled is much much lower than uninjected control and miRNA injected embryos.