I have cloned the 3'UTR of my gene in the pmirGLO vector from Promega and using Neuro2A cells for co-transfecting the GLO-3'UTR plasmid (100ng) and miRNA mimics and inhibitors (50nM each). Surprisingly, in every set I am getting low readings for the Renilla luciferase (endogenous/transfection control) in mimic or inhibitor transfected cells as compared to cells transfected with scramble. That's why any decrease in the firefly reporter reading is getting non-significant after normalizing with the respective renilla. I have tried to adjust the transfection protocol by trying to play with the reagents, for eg. Lipofectamine 2000 (0.5-1ul), GLO-3'UTR plasmid (100-150ng), mim/inb (50-100nM), but the results are the same, i.e, 'decrease in firefly as well as decrease in renilla for mim/inb'. I have used Dual Luciferase reporter assay kit as well as Dual Glo luciferase assay kit (Promega) within 24-72hrs time point after transfection.

Having said this, I have also tried to do the assay in Neuro2A cells over-expressing our miRNA of interest; to avoid all the errors arising due to co-transfection. But there also I am seeing a repeated decrease in the reading for the Renilla as compared to Scr over-expressing cells. This is very strange. Why would the renilla be affected by the presence or absence of my miRNA? And to avoid pipetting errors I prepare a master mix of transfection reagent and plasmids, from where I add to the different wells (96 well format). If anybody can shed any light on this it will be very helpful