Request for advice — dual-luciferase assay setup. PLEASE HELP
Hey everyone! I am desperate for help. I've also sent out these questions to trusted members of my committee and general support peeps, but I'm trying to cover all bases. Please let me know if you have any experience with this! You're so appreciated.
My setup: a bi-cistronic firefly–IRES–Renilla reporter (the RELITE system from Vitale and D'Agostino et al. 2025) used to screen Sec61 translocation inhibitors. We transfect HEK293 cells with X-tremeGENE, dose compounds in 96-well format, and read white opaque plates on a BioTek Synergy H1. We use a homemade luciferase reagent rather than a commercial dual-reporter kit.
We have tried altering many of the parameters, including making the reagents more concentrated, increasing transfection time, completing the assay in the dark, etc. We are trying to narrow down the issue of signal, and I have some questions for you all if I could get some input.
- Background signal. What level of background do you typically see, and how do you distinguish it from low true signal? Any advice on blank wells, untransfected controls, or reagent-only readings would be helpful.
- Transfection efficiency. How do you check it, and what do you consider acceptable before trusting the ratio data?
- Instrument suitability. Based on your experience, would a Synergy H1 be adequate for this assay, or would you recommend a different reader?
I am getting signal in my nontransfected cells that are treated, I am getting signal in my nontransfected cells that don't have treatment in them. I have high background signal in my blanks.
How should I go about testing for transfection? I am so careful about ensuring there is NO DNA in my nontransfected wells, I am so confused by this result. Why is the fricking signal the same? Are they just both not transfected? Did I accidentally get a transfected HEK293 cell line? (Borrowed the cells from another lab that also does dual-luciferase assays.)
My signal also has high variability. My SDV is broad. I had so much deviation its a joke. I literally TAKE PHOTOS of my wells to ensure they are all even at every step. I'm looking at my multichannel tips as I take up liquid. My pipetter is old, so sometimes a random tip doesn't pull, and I will GO BACK WITH A P200 AND INDIVIDUALLY PIPETTE THAT WELL TO ENSURE IT IS EVEN. I fricking refill the tip boxes with tips I know seal best because I want to mitigate error. I've been in labs for almost 10 years. Do I suck? Is it my machine? Is it me?
This assay is VITAL to my dissertation and first author publication(s). PLEASE help if you can. THANK YOU! <3