I'm co-transfecting 1) a reporter plasmid with an inserted enhancer and the luciferase gene and 2) a plasmid with the Egr-1 transcription factor gene using jetPRIME reagent.
I'm wondering what would be the best ratio to co-transfect these for SH-SY5Y neuroblastoma cells into 24-well plates in order to get the highest expression efficiency. My current thought is to transfect the plasmids at 1:1 pmol, but I was wondering if anyone has done previous work with transcription factors and if I should add more or less of the Egr-1 plasmid?
For reference...
the jetPRIME protocol states to add no more than 500ng of total DNA per well for transfection
(I've attached the jetPRIME protocol)
size of luciferase reporter plasmid: 3937bp
size of Egr-1 transcription factor plasmid: 7099bp
How are you going to normalize your data? Do you have an internal transfection efficiency control (e.g Renilla luciferase driven by a constitutive promoter)?
As for the ratio, would try proportionally less of the transcription factor expressing plasmid to mimick cellular conditions better (many copies of mRNA per copy of chromatin).
Note that neuronal cells are typically all but impossible to transfect...hopefully your neuroblastoma cells/jetPRIME combo will work...if not, consider viral delivery.
Not ideal. Correcting for protein content does not factor in transfection efficiency very well; better than nothing I guess but typically not sufficient for publication anymore. You should also try dose responses with your transcription factor plasmid (instead of simply with and without). Also your reporter plasmid contains a promoter I assume, not just an enhancer?
Sébastien Soubeyrand Thank you, that's good to know. I've previously tried a dual-luciferase assay using Renilla as my normalisation control vector and the Renilla values were spread out and inconsistent, so that's why I switched to normalising with a BCA assay. Trying with dose responses is a good point, I will do that as well.
To clarify my "luciferase plasmid" is the CpG free lucia luciferase which is secreted into the cell media so I've been using the QUANTI-Luc GOLD kit (InvivoGen) for luminescence detection. And when I had transfected the cells with Renilla, I was analysing the cell lysates with Stop N Glo reagent from Promega.
And yes the luciferase reporter plasmid contains a promoter.
If you used dual in the past, did you get high values (even using 1-2% vs firefly; assuming a strong SV40 or equivalent promoter)? You should have. If not, your transfection efficiency is probably quite low and could explain your inconsistent values. Another big one is the lysis buffer used; the Promega PLB has been optimized and is a must for this assay to work properly.
Otherwise, it should work pretty well with lucia but again, normalization to protein content is very 1990s. That being said, if you perform your dose response, it may help (make sure you always use the same amount of total DNA across; use stuffer DNA if needed).
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