housekeeping gene rtpcr

Bill Alexander alexanderw at cber.cber.fda.gov
Fri Feb 2 13:36:47 EST 1996


In Article <4es6op$kaq at damage.usa1.net>, suihuang at usa1.com
(suihuang at usa1.com) wrote:
>In article <alexanderw.1173512937B at newssrv.dcrt.nih.gov>, 
>alexanderw at cber.cber.fda.gov says...
>>
>>In Article <4eo69l$69d at news.bu.edu>, anttro at bu.edu (Anthony Trombino) wrote:
>>>We are doing RTPCR using GADPH as a control, and were are finding that even
>>>with equal amounts of rna I am getting varying pcr products of gadph.
>>>Any suggestions? Are housekeeping genes constantly expressed even after
>>>stimulation of cell
>>
>>Always make your own standard.
>>Regards,
>>
>>Bill Alexander
>>alexanderw at cber.cber.fda.gov
>>
>
>
>The original question by Anthony T. refers to the use of INTERNAL standarts in 
>COMPARATIVE RT-PCR, therefore it doesn't make much sense to use "self-made 
>standarts" as in COMPETITIVE PCR.

It doesn't make any sense to use a standard that varies as a standard to
measure expression with.  (People don't use the size of the King's foot as a
standard of measure any more.)  

Why do so many scientists believe in the stability of expression of
housekeeping genes?  Is it so much less work if they do exist?  The problem
of using different primers for the amplification of your standard should not
be overlooked.  This would not be acceptable at The US Patent Office (even
if you got it published) unless you have all of the controls showing that
the standard amplifies at the same rate.  

You still should be proving the consistency of your "standard gene" in your
experiment.  If you are doing PCR anyway why not just make the standard(s)
yourself?

>
>As for the GADPH as a control: this gene is not very suitable as its expression

>is highly growth dependent. furthermore, there are many retro-pseudogenes of 
>GADPH which might be amplified if you have DNA contamoination in your RT.
>similarly the widely used beta-actin is also expressed with some growth- (cell 
>cycle-) dependency.
>I used beta-microglobulin which seems to be quite growth independent 
>and to show constant expression levels. (beta-microglobulin is expressed 
>constitutively in every nucleated cell. however it is induced by some
cytokines).

Did beta-microglobulin amplify at the same rate as your experimental gene?
How did you prove this?

>
>Hope this info might help. good luck,
>
>Sui
>Children's Hospital Boston
>
Regards,

Bill Alexander
alexanderw at cber.cber.fda.gov

     "640K ought to be enough for anybody." -- Bill Gates, 1981



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