I'm doing luciferase assay to determine microRNA regulate one target gene expression. The empty vector is pmiRGLO, wild type is pmiRGLO with 3' UTR of the target gene, mutation is vector with seed region deleted 3' UTR. I used HEK293 and CHO cells for this but all got very low firefly values of WT transfected samples. Firefly values of empty vector and mutation were hundred folds higher than WT ones. I've tried many times and I really don't know why. Does anyone has suggestions? Thank you so much for your time.
Well firstly, I believe you are achieving the question you've set out to answer.
Your Wildtype binding site is being regulated by the miRNA, therefore the luciferin mRNA is being highly downregulated and therefore its yielding very low firefly values.
Furthermore your empty vector and mutant binding site will not be regulated by your miRNA of interest and therefore the luciferin mRNA will continue to translate into the protein form and yield high values.
I do however think your fold change is drastically high. How long do you culture your cells following transfection before performing the luciferase assay? Also do you use a dual luciferase plasmid which has firefly and renilla because then you have a control within your experiment that normalizes all your repeats and experimental groups. If not, how do you determine all your transfection efficiency and normalize your data against each other?
Matthew de Cruz Thank you for your answer. I understand that the luciferin mRNA should be downregulated by miRNA. The problem here is even in the miRNA control transfected cells, firefly values are super low compared to vector and mutation groups. I'm sorry I didn't mention in the question that I do use a dual luciferase plasmid. However, the values of renilla don't have big difference. So the ratio of firefly and renilla between WT groups and others are about 100 fold difference. I did the assay 24 h after transfection.
Sorry for the delay, so I would check the settings on your plate reader. I previously had an issue where it was just measuring the surface of my wells and that gave off very low values and then someone showed me a setting in which the plate reader measured through the wells in my plate and gave off nice high values.
I don't work at the institute where I did my luciferase assays so I can't go remind myself at what settings I used to get accurate values. But if you have someone in your department that uses the plate reader frequently, they might know how to adjust your settings for your experiment. Hope it works, good luck.
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