Trouble with GAPDH housekeeping gene - (Aug/14/2009 )
I'm using PC12 and HEK cells that are transfected with CB1 receptors and i'm doing PCR and gels for them.
Before i do PCR with CB1 primers, ive checked with GAPDH primers first but i find that my GAPDH is only working for HEK and not PC12 cells (only HEK cells are showing bands ). So i used another house keeping gene (beta actin) and then CB1 primers , and this time both PC12 and HEK cells worked. I've done this twice now.
Does anyone know why GAPDH is not showing a band for PC12 cells?? 
Thanks
Hi there!
Very obvious question, but did you check if your primers can bind to the GAPDH seqeunce in PC12? HEK are from human origin and PC12 cells are derived from rat...so there might be differences regarding the GAPDH sequence and then the PCR doen't work...
Stardust
stardust on Aug 17 2009, 12:25 AM said:
Very obvious question, but did you check if your primers can bind to the GAPDH seqeunce in PC12? HEK are from human origin and PC12 cells are derived from rat...so there might be differences regarding the GAPDH sequence and then the PCR doen't work...
Stardust
You're very right! I didnt realise PC12 was rat and HEK human, after i checked on sequencher, my GAPDH primers werent matching very well with the rat sequence
Cheers!
No problemo!