I want to transfect miR-145 MIMIC to cell. the target sequence of miR-145 is inserted to a vector and is transfected to this cell. indeed, i want to transfect micro RNA mimic and target to cell and evaluate the effect of this miR. The use of positive and negative micro RNA controls are necessary for my study?
Dear Mohammad Shayesteh
Generally, in the basic studies you need negative control to evaluate the effect your experimental group (for example micro RNA). While, in the case you want to compare your agent with standard agent, it is better to add positive control in your study. In addition, editors of top journals ask you to add the negative and positive control for the best interpretation. Hence, as an editor of the medical journal; I recommended to you concerning this issue.
I hope that I could to help you
What if the process of introducing the gene (via transfection/transduction) results in gene expression changes? Then how will you know it's the transfection process or the miRNA vector that you've inserted causing this effect.
Hence why you need to show that a scrambled/non-mamallian (i.e LacZ) vector is necessary to show that it's the target miRNA vector that you've inserted that's causing the gene change and not the process of transfection. You will need minimal of a negative control to rule out procedure-related changes. Negative control is the equivalent of a placebo control.
Hello,
It is important to provide a negative assay control, for all reasons stated above (to show that it is the miRNA of interest causing change, not the vector). In this case, the negative control would be an empty plasmid vector. When transfecting cells with miR-124a, I used PCMV-miR (mouse) as an assay control.
Hope I could help!
Indrani Das
Hi Mohamed,
I think that you will use the dual luciferase assay, so first, you don't need positive control but if you want one choose a pair of microRNA/target from bibliography. Secondely, for negative control you must use an empty vector and the vector with the insert of your target but without mimicomme microRNA, cause you will work on cell-line and they have their own microRNAs and riboproteins.
Good luck.
Amine
Of course, you need negative control for miRNA experiment.
In addition, you should prepare mutated target sequence to know specificity of miR-145.
Hisashi
You absolutely must start with a negative control in order to be able to assess if the miRNA is having an effect. If you see an effect, then you will want to include additional controls as others have suggested (i.e., mutagenized target sequence). The good thing about a dual-luciferase assay (if that is what you are doing) is that it includes an internal transfection control (the luciferase that does not have your target sequence attached), so that is one less control you need to worry about. If you want a positive control, consider generating a vector with multiple repeats of the miRNA binding sequence in the 3'UTR of the reporter, and co-transfecting that construct with your miRNA mimic. If the miRNA is functioning properly, it should dramatically decrease the reporter expression.
All the controls are essential for publishable results, but start with the basic negative control first to make sure you have something worth pursuing before you go and start constructing other vectors.
Hi, The subject of my doctoral thesis is related to microRNA suppression.
Can I use a negative micro-RNA or a mock vector as test control in the Luciferase assay? Or should I use it (psiCHEEK2-GPDH)? May you guide me.
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