I'm doing luciferase assay in transfected cells with two differents alleles of a polymorphism. Both alleles are cotransfected with my microRNA from interest and a microRNA negative control. One allele has "seed site" from this microRNA and the other allele don't.
I found differente expression of my gene comparing the alleles + miRNA, but what I can not understand why my negative control miRNA is keeping different in the alleles comparison.
It should be the same gene expression in both alleles cotransfected with negative control, right?
Anyone does have an idea what could be happening?
Thanks a lot.
Hi Juliana
Could you please tell me which cell line are you using for transfection and what miRNA is it? It is possible that those cells may be expressing fairly good amounts of the miRNA of your interest, which is resulting in differential results even with the miRNA control. So when you co-transfect miRNA control with either of the alleles, the allele that contains the seed sequence would ideally be repressed if your cells contain endogenous expression of that miRNA as compared to the allele which does not contain the seed sequence.
Hope it helps.
Kiran
Hi Kirandeep! Thanks a lot for your answer.
I'm using FaDu cell line, a cell line from farynx cancer and microRNA miR-4421 (mimics mirVana) .
How can I know it? Maybe instead of using negative control I should use a mir-4421 inhibitor?
Thank you!!
http://amp.pharm.mssm.edu/Harmonizome/gene_set/FADU/Klijn+et+al.,+Nat.+Biotechnol.,+2015+Cell+Line+Gene+CNV+Profiles
Follow this link. Just type mir4421 in the filter. It shows that miR-4421 is expressed in FaDu cell line.
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