Hi all,
does someone have experience with studying the interaction of microRNA (miRNA) and its target sequence cloned into a Luciferase Reporter vector? Actually using pGl3-Basic vector from Promega but the miRNAs which are co-transfected appear to have rather high effect on the pGl3 Backbone. The inhibition of the inserted 3´UTR is rather negligible. Do you know or even use a Luciferase Reporter Plasmid where miRNA has the least Backbone interaction so the cloned sequence (either promoter or 3´UTR) can easily be studied?
Happy for any comments or suggestions!
Take care
Hi Stefan,
I would recommend you pmirGLO dual luciferase reporter (https://www.promega.es/products/reporter-assays-and-transfection/reporter-vectors-and-cell-lines/pmirglo-dual-luciferase-mirna-target-expression-vector/?catNum=E1330).
It is a specific vector for 3' UTR cloning and miRNA targets, you can either perform a standard cloning and insert your region of interest or simply the miRNA target using the default protocol that they propose.
I have used it last month and the results were pretty good. My only recommendation would be not to use restriction enzymes whose targets are too close in the MCS or they won't work properly...
Hello Mauro,
happy about your answer. Did you check for any miRNA mimic and pmirGLO backbone effects?
Hi again Stefan,
Our experiment was pretty simple. We were checking for native miRNA inteaction, so we transfected:
- Positive control => Vector
-Negative control => no vector (the vector has a strong promotor, so you always get luciferase activity, in the original design we were going to also use an effective miRNA as miRNA interaction control)
- Vector + insert with a mutation
- Vector + wild type insert
At least in the COS1 cells that we were using, I did not detect any mimic or backbone effect, the background luminiscence was low in the negative controls, and in the end we were able to detect a significant reduction in the mutated insert while WT and the positive control showed almost the same values.
I hope you find the information useful, but I am still doubting if I answered what you asked.
Best regards
Can you find seed match for your miR in the plasmid? If not than the effect is likely indirect, maybe your miR inhibit a transcription factor important for the promoter you use. If you have a seed match you may be able to mutate it.
Hi Yoav,
this is helpful and I´ve looked for seed matches in the Plasmid Backbone before. Unfortunately, there are >1 seed matches which might complicate mutation studies. We´re thinking about changing the luciferase reporter plasmid, but before we do I´ll check it for miRNA interaction.
Thanks to you and also Mauro!
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