So I am trying to use Luc mRNA to transfect 293T cells to measure the transfection efficiency of different nanoparticles in 96 well plates. However, repeatability of my testing is not quite well (even I have done experiment using one batch of cells).
I think the reading of luciferase are affected by the density of cells. One of thinking is to normalize cells number using BCA. What I am thinking is that after Luc reading, I will remove all medium and directly adding ripa to lysate cells and use BCA to detect the protein concentration of 96 well plates to normalize luc reading. Dose this idea sounds reasonable. Is there any other better solution? Thanks!
When transfecting using plasmids, a control plasmid is normally co-transfected to normalize data, but if this is not possible in your design, protein quantification will provide some normalization and should improve your results.
You think the density of cells is affecting your transfection... at what confluency are you transfecting? Are seeding the same number of cell in each well and between your experiment? Try using low passage HEK.
Thanks for your reply! So I think the confluency should be ~70%. Typically I seed 10000 cells per 96 wells.
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