Chosing the best housekeeping gene! - (Jun/01/2009 )
I'm trying to detect the level of expression for two different mRNA with real-time PCR (SYBR green) during apoptosis. I'm using GAPDH and b-actin as my control.
But I need to change both my housekeeping genes primers as they start to show downregulation during apoptosis.
I'm looking for a substitute for these two genes. Do you think 18S would stay constant during apoptosis?
Ideally you will screen a few (5 or more) housekeeping genes and then choose the ones that are most stable. You could try B2M or HPRT.
PMID 14748434 (search this number in PubMed) found that GAPDH was the best housekeeping gene during apoptosis. Also B2M was promising, while 18S was not... I don't know, maybe your cells are different from theirs? But I agree with bob1, best way is to check yourself.
ok, thank you guys.
so I'm gonna design new primers for 18s, B2M and HPRT
make sure to check ribosomal proteins as well... my favorite is Rpl13! Best ever, trust me. I've tried a lot.
Hello! I'm facing the same problem.. both GAPDH and b-actin are affected during apoptosis... so, I would like to ask which House keeping primer worked finally ?? Please help me ...