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Chosing the best housekeeping gene! - (May/31/2009 )

Hello all,

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?

-Curtis-

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.

-bob1-

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.
Cheers,
Minna

-Minna-

ok, thank you guys.

so I'm gonna design new primers for 18s, B2M and HPRT

-Curtis-

make sure to check ribosomal proteins as well... my favorite is Rpl13! Best ever, trust me. I've tried a lot.
Cheers,
Minna

-Minna-

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 ...

-Star*-