induction estrogen responsive gene (PS2 & PgR) in MCF7 - (Jun/27/2006 )
I'm trying to work out the conditions to induce the expression of estrogen responsive genes in MCF7 cells.
The two conventional genes I'm trying to use are PS2 and progesterone receptor.
I grew the MCF7 cells in charcoal stripped serum in phenol red free medium for 10 days then I added 10-8 estrogen but at early time points I still have strong expression of these genes.
In other papers, they always have a nice increase in expression with time (with no expression of PS2/pgr at early time points).
What am I doing wrong?
Did you get the cells from the people that published the paper? That could make a difference, I think
No, other labs have used MCF7 in the same purpose and seem to work.
I was using 10% charcoal stripped serum/phenol red free medium (FCS-CSS). It is possible that my medium still contains some low level of estrogen and growth factor.
I have been advised to plate my cells in 10% FCS-CSS but after change to lower FCS-CSS (up to 1%).
I'll try that...fingers crossed.
I got the problem sorted.
I starved my cells for several days and progressively reduce the serum in the medium 10%-1%.
I think my main problem was the method of detection. I started with standard PCR while most results to look at PS2 expression were done in northen blot.
I used real time PCR and i got really good results.
Cheers for the input!