Hi Olivier,

I work on HEK-T (5-30 passages) cells for this experiment. Do you perform multiple transfections in different experimental samples on top of Firefly and Renilla Luc. constructs? If so, do you keep the same amount of total DNA introduced to the cells for transfection? 

Hi Gonca,

We use the Promega DLR kit with ref: E1910. It works very well and can be used for multiple types of experiments. Are you using a different kit?

If your DLR experiments have a high variation regardless of experimetal trials and cell types and you just transiently co-transfect separate plasmids to express FL or RL in cells, it'll be good to adopt dual reporter system (e.g, two independent promoters in the same plasmid). It could reduce a variation from different transfection efficiency. Otherwise, uneven lysis during assay or irregular spreading of cells on a culture dish before transfection could also give a high variation.

Dear Gonca Bayraktar,

We used the kit Promega DLR con ref: E1910,  with good results. Best regards.

Thanks for the answers. @Duane and Daniel: I'm also using this kit Promega. The use is quite simple and straightforward.

I work with a special vector that is CpG free. I generally get consistent reading from my Firefly Luc. which I obtain with this construct however my Renilla Luc. reading varies a lot. I also tried a random Renilla Luc. vector under SV40 promoter that people use for the normalisation of Firefly reading however it was also not consistent. How do you collect and lyse the samples using the kit from Promega? As suggested to wash once with PBS, I aspirate the medium first, then slowly add PBS and then aspirate it and add the Passive lysis buffer immediately, then scrape cells and collect. I give 2x times freezing/thawing using liquid nitrogen and thats it. I dont know where the inconsistency comes from. Even if I prepare the transfection mixture in the same tube and distribute the readout may vary. Ans also when Renilla is expressed with some other constructs (I did a test) although the same amount of Renilla DNA is used, readout varies..

We stabilised it to a better extent by using HEKT cells plated in 96 well-plate since this eliminate the need of lysate being collected in a tube and then placed back in a 96 wp for the readout. Another thing we also did to clone Renilla into the identical vector backbone that I was using with my in vitro methylated construct. Renilla readout is still somewhat more varying compared to the Firefly, but at least these few tricks improved the assay.