Hi guys! I did real time RT-PCR to quantify gene expression ( caspase8, bcL2 and bax). MCF-7 cells were treated with combination of tamoxifen and rHuEPO, RNA extracted, reverse transcriped and subjected for PCR for target genes. All genes amplified well however this amplification in treated cells is not significantly different from untreated cells any explanation for this as this combination expected to change these genes?
Thank you Anastassia, that's possible just wondering as tamoxifen stored properly in powder form even it is new and as you know it can produce effect even in a very small amount, I used it in 10ug/ml.
Did you see your cells dying before you harvested them? If they didn't look sick then I agree your treatment probably didn't work. You could also run a western and look for cleaved caspase 3 / cleaved Parp to confirm cell death.
If you do see cell death, but still see no significant differences in expression of these genes... I am not 100% sure you would expect to see mRNA level changes under these conditions - all these proteins will become active (caspase 8 will be processed, Bcl-2 will be inhibited by the BH3 proteins freeing up Bax to form pores in the mitochondrial outer membrane) during apoptosis, but their overall levels might not necessarily change at all. I think you'd expect more of a change in RNA levels of the BH3 proteins (Bim, puma, noxa etc) as these are often transcribed under conditions of cell stress to promote apoptosis.
- Badges
- Science topic