Hi guys,
I've been struggling with this issue for close to three months now.
My issue:
I transfect my PC-3 cell line with Lipofectamine 2000 and my siRNA (PLK1) and with the controls non-targeting siRNA & Lipofectamine, Lipofectamine only and Naked siRNA. I use a 24 well plate, each sample has a triplicate - so my transfection is set up with triplicate amounts and 200ul of the tranfection reagent added to each. So that means 0.76ul of siRNA, 1.2ul of Lipofectamine and then 198.04ul of Opti to make 200ul per well. These 200ul are then added to 300ul of Opti-MEM which is already in the plate.
I'm having issue with cell number, sometimes really full plates and sometimes not, and now having problems with obtaining a suitable RNA concentration, predominantly below 100 ng/ul.
I have already increased the incubation periods from 24hr to 48hr and 72 hr - results haven't been obtained yet as the assays are currently running.
Now the next problem is that my MTT assay is also not working properly. For this I'm simply using lipofectamine and lipofectamine & non-targeteing siRNA as practice in RPMI. Lipofectamine is bad for cells- we all know this - but I'm obtaining % viability well over 100%.
I'm completely lost, so any help/recommendations are greatly appreciated.
The way you've phrased your question(s) is a bit confusing - you can't obtain cell viability greater than 100%. If your issue is that your cells are dying, you obviously need to lower the concentration of transfection reagent, whereas if you are having problems with the efficacy of transfection, it's probably an issue with your reagent. Generics aren't tailored to specialized cell lines like PC-3, but there are kits out there designed for prostate cancer cell lines (see https://altogen.com/product/pc-3-transfection-reagent-prostate-cancer-cells/). If you're trying to model concentrations after a protocol, look there to see if your starting amounts and procedures match up. If not, there's a clue to your dilemma. Good luck!
Dear Ted Bremmer,
Apologies. Basically I have two issues which kind of go hand-in-hand.
I'm setting up my 24-well plate and my MTT at the same time, because I'm testing the nanoparticles in both. I know I can't (or shouldn't be) getting a cell viability over 100% but I am in my MTT. I'm unsure whether it is the lipofectamine or whether I'm doing something wrong prior. I have attached some sample data below for you to see.
It's not the transfection itself that is causing the issue - it's the lipofectamine samples which are causing some head scratching.
I will have a look at the link you sent also.
Thanks for your help
PC-3 cells are okay to transfection normally, but sometimes it happens.
You could give a try on "resetting" the doubling time but that is quite toxic and not an easy method.
Did you coat the plate, maybe cells are not so well attached?
Finally, other than MTT, did you try Alamarblue, Prestoblue, Crystal violet and so on to assay the toxicity/viability?
I used PC-3 cells with L2000 and Alamarblue and it work quite well in my case:
Article Stereoselective pH Responsive Peptide Dendrimers for siRNA T...
Good luck :-)
- Badges
- Science method