When I transfect HeLa cells with lipofectamine 2000, cell size of 10-15% population increases within 48hrs. Transfection works quite well in our lab but I could not see the depletion of the gene. Why does it happen?
Hello! It's very hard to give some help. Please, can you tell us more details.
What do you mean exactly by "cell size of 10-15% population increases"? Do the cells really change of aspect? How big is the size increase?
You say that transfection works good in the lab... does it mean that your labmates manage to transfect the same cells and you are the only one who has issues?
"depletion of the gene", do you mean that you tried to transfect siRNA to silence a gene? do you have controls?
Looking forward,
Bests, Sandra
Thanks for your reply Sandra
Transfection works well in the lab means we frequently do the transfection with lipofectamine 2000 in 4-5 cell lines and we get app.50% depletion for most of the genes. But suddenly, the whole lab is facing the problem of cell death, increased cell size and almost no depletion in spite of the fact that we used new freeze down, two lipofectamine vial of two different batches and the optiMEM as well. Lipofectamine which we have used earlier and transfection was fine, is giving the same result. We do three days transfection in control siRNA sample as well. I am attaching the images of control cells. You can find the difference in the cell size.
Using the eyes, It is hard to detect "10-15%" size changes from those images.
Antibiotics are not present in the incubation media? I also know that at certain levels, L2K becomes cytotoxic.
What is your overall transfection effciency? If you are not getting around 70-80% total transfection you cannnot reliably quantify gene knockdown using PCR.
http://www.sabiosciences.com/validaternai.pdf
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