PGL3, miRNA inhibitor co-transfection question - (Dec/10/2007 )
I have another question.... I am trying to transfect PGL3, Renilla and Dharmacon's miRNA inhibitor. What is a good transfection reagent to use?
I normally use Lipofectamine for PGL3, Renilla and a vector encoding miRNA. I wasn't sure if I could use Lipofectamine for my current experiment, because of the size of miRNA inhibitor (approx 23 bp). Does anyone have any experience with co-transfection reagent for miRNA inhibitors?
I appreciate your suggestions....
We use lipofectamine 2k to transfect 3 things: 2 plasmid and a miR. It works. You can also try two step transfection: first transfect the reporter vectors, 24 hrs later, split the cells and transfect miR.
we use lipofectamin 2k as well, and it works well for all of the plasmid you've mentioned.
Is it ok to transfect the miRNA first, after 24hours removing the medium only,then transfect with plasmid ?
For 2-step transfection, either way should be OK. The reason that we transfect plasmid first is because after the plasmid transfection we split the cells for different miRNA transfections so that the plasmid transfection efficiency is consistent. One step transfection should also be fine. In one step transfection we transfect two plasmid (pGL3, pRL-TK) and a miRNA and obtain expected result.
It also depends on the purpose of your experiment. If you want to look into whether the miR targets the reporter, then why not first introduce the target into the cell and then transfected the attacker?
hi, thanks, yes, I want to look into whether miR targets the reporter, but I have two UTR target sites constructs and two mutant site (target sites were mutated) constructs, I am not sure whether after splitting cells the cell number between the groups can still keep the consistency. I also think it is better to first introduce the target into the cell and then transfected the attacker, Do you have any suggestions and solutions?