I'm going to transiently transfect subcutaneous pre-adipocytes with His-tagged plasmid DNA [pcDNA3.1(+)] containing ADIPOQ gene with a SNP to study the adiponectin expression. Can any expert shares to me what is the best lipid based transfection reagent to transfect pre-adipocytes; FuGENE 4K or Lipofectamine 3000? How long is the most optimum waiting time after the transfection process to proceed with protein expression? I'm also going to treat the cells with few drugs, and will perform RNA quantification (qPCR) and measure the level of protein (adiponectin) via ELISA. Should I perform western blot as an analysis to confirm protein expression before I proceed with drugs treatment after the transfection done? Million thanks in advance for the ideas and generous support.
Ideally you want to wait at least 3-6 hours after transfection. You should do a selection step after transfection before you do your expression. The plasmid you are using has a marker gene for G418 selection so any surviving cells will be successfully transfected.
You definitely should at least do a qPCR to confirm that your gene is being expressed although a western blot would be better. Definitely confirm expression before your drug trial.
I think Lipofectamine 3000 works fine and can be used for your transfection purpose, since you will be transiently transfecting your cells your protein expression can be seen after 6-8 hours but it is recommended to wait for 24 hours and then proceed with your further experiments. What I would suggest is you can transfect more number of cells and then divide them into three parts, one part you can use for your qPCR assay, one for protein expression study and one for your drug trial experiments.
Million thanks Pankaj Sharma & Robert Adolf Brinzer for the suggestions.
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