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Chosing the best housekeeping gene!


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5 replies to this topic

#1 Curtis

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Posted 31 May 2009 - 10:00 PM

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?

#2 bob1

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Posted 02 June 2009 - 04:32 PM

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.

#3 Rsm

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Posted 02 June 2009 - 05:36 PM

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
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#4 Curtis

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Posted 02 June 2009 - 08:10 PM

ok, thank you guys.

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

#5 Rsm

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Posted 04 June 2009 - 01:26 AM

make sure to check ribosomal proteins as well... my favorite is Rpl13! Best ever, trust me. I've tried a lot.
Cheers,
Minna
I got soul, but I'm not a soldier

#6 Star*

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Posted 16 June 2009 - 06:05 AM

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 ...




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