Goodevening!

I am currently optimizing stimulation time for RANKL induced RAW-Osteoclast for qPCR analysis.

for osteclast markers such as TRAP, MMP-9, Cathepsin K, a significant mRNA fold increase (about 40fold increase and above) was observed with RANKL stimulation of 2 days.

however, for osteoclast transcription factors such as NFATc1, and cfos, the fold increase was very minimal (about 1.02) or even less than the control group.

stimulating RAW cells with RANKL 50ng/mL for 3 days or 4 days did increased cfos transcription level by 1.2.

however, I can't see any increase of NFATc1.

Has anyone of you had experienced similar problem?

here is my protocol.

seeding RAW 264.7 cell in 6 well. 2 x10^4 cell/ml 2mL/well

culture for 24 hour.

then refresh media. stimulate with RANKL (50ng/mL final concentration)

for 2,3, or 4 days.

media was refreshed every 2 days

on day 4 , I have seen few multinucleated osteoclasts.

should i change the media in to absolutly phenol red free media? or re-design my primer? the melt curve was very clean though...