reproducibility of luciferase assay - (Oct/24/2008 )
First of all, I am a chemistry guy and I worked on transfection for half year. I faced problem in reproducibility. (well, I faced it in MTT assay too) I hope someone can help me. THX!
I synthesized some cationic polymers and wanna to test for its transfection efficiency and I used luciferase assay to quantify it. The RLU are always different a lot, even with transfection kit (+ve control).
I use 96 well plate with 5x10^3 cell/well and look for 48 hr expression, triplicate, reagents were mixed 3.5 times and 1 portion added to wells. I worked on 2 96 well plates in a time, cell lines were U87 and U373.
reply if anyone can help!
You will find something interesting in JALA (Journal of Association for Laboratory Automation)
some years ago they issued two good tutorials:
1) Assay Optimization: A Statistical Design of Experiments Approach (doi:10.1016/j.jala.2005.11.001)
2) Efficient Assay Development and Screening at a Small Research Site (doi:10.1016/j.jala.2006.02.001)