Hello,
I've cloned a 3' UTR of interest into a pGL3 firefly luciferase vector and have transfected it, along with the pRL-TK Renilla luciferase vector control (1:10 ratio) and my miRNA of interest, into kidney epithelial cells using Lipfectamine 3000 for 48 h. After using the Dual Luciferase Assay from Promega, however, I am getting luminescence values from 300 to 1200 for firefly luciferase and 15-30 for Renilla luciferase (background is 12). These values seem incredibly low but my only frame of reference is Cell Titer Glo, where I get luminescence values of ~50,000. Also, I have high variability among replicate wells. Is this a sign that I have super low transfection efficiency? Or something else?
I have had no issues transfecting the miRNA mimic alone. Also, I've sequence the portion of my plasmid where I've cloned in the 3' UTR, but not the entire plasmid. Is it possible my plasmid could be screwed up?
I'm new to all this cloning/plasmid stuff and would appreciate the help!
Thanks,
Cory
Hi Cory it may be the sensitivity of your equipment can I ask what make and model of plate reader you are using.
Hello Jamie, thanks for the question! We have a Synergy H1 by BioTek. I have been able to detect Cell Titer Glo signal without any problem (~50,000 RLU), and I would think there should be no difference with the Dual Luciferase Assay...
Hi Cory
there can be differences in signal between plate readers as I operate the BMG Pherastar for CMDS at Birmingham. We have seen differences in sensitivities between monochromator-based multi-mode microplate readers when compared with filter-based optical module plate readers (much more sensitive). The Synergy H1 by BioTek can be turned into a high-performance hybrid system with the addition of a filter-based optical module so do check that you have this. However I would contact Bioteck as they may have some details about the two assays and how to tweek the assay conditions to get the best readings.
Thank you so much! These are all great ideas! I've been wondering if it's a plate reader thing since the Dual Luciferase Assay boasts their ability to detect extremely low signals. Thanks for your service here on researchgate. I can't thank you enough.
-Cory
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