In our lab we are wondering if we can check the effect of silencing a gene on a specific regulatory sequence which we have cloned in a reporter luciferase vector. I know taht plasmids can be nucleofected employing amaxa nucleofector kits as siRNAs, but I am not sure if we nucleofect both (luciferase vector plus siRNA) the efficiency would be affected or not... Does anybody have any experience doing it? Thank you!!
It really depends on your experimental design:
You can nucleofect both (siRNA and a reporter) and see an immediate effect of siRNA on gene expression. Or you could introduce reporter first, let it express for 24 hours and then add siRNA. In this case you would have a timed effect of your siRNA. Generally i would recommend to introduce reporter first. You can do it in balk and have large number of cells with similar level of the reporter expression ready for siRNA experiment.
Also using destabilized luciferase helps a lot.
The nuclefection will increase efficiency of the siRNA transduction. However you could also use a more traditional transfections reagents.
Option1: Transfect siRNA into cells first with tranditional chemical reagents or nucleofect on Day 1 (PM), the next day cells will stick to dish, which is good for luciferase construct transfection with tranditional reagents. It is better to transfect siRNA first to see knockdown effect.
Option 2: Mix siRNA:luciferase as 10:1 or more, and then nucleofect them at the same time. Luciferase is very sensitive so don't worry about the expression level.
Thank you for the responses. In this case, we are using suspension cell cultures. I was thinking of nucleofecting the siRNA before or both siRNA and luciferase plasmid at the same time as you commented Xianfeng. I guess it is better for the survival of the cells doing one nucleofection with both, but I am wondering if the measurements are consistent; I mean, if the relative efficiencies of siRNA and plasmid remain constant among samples... Xianfeng have you perfomed it before?
Yes, I did it before (option 2). But I don't like nuclefection because of the death of many cells. If you worry about the consistence, do 4 duplicate at least, even 6.
If your cells are suspension cells, nucleofection may be better. Most chemical transfection reagents don't work well for suspension cells.
- Badges
- Science method