"Standard" housekeeping gene

darin at lenti.med.umn.edu darin at lenti.med.umn.edu
Sat Jun 25 13:46:58 EST 1994


I'll put in my $0.02 worth.  I strongly feel that actin, GAPDH etc have 
been misused as a control for RT-PCR in that often times the no RT 
control is left out.  This is especially a problem with GAPDH which has 
200 retropseudogenes in mouse.  I think the HK gene control without the 
no RT control is completely useless in most cases.  Actin suffers similar 
problems.  
	I am currently using porphobilinogen deaminase as a control for RT-PCR.
  I think it is reliable as it is expressed ubiquitously, has no 
retropseudogenes (at least in mouse), is constitutively regulated (as far 
as I know), and is probably expressed at levels 
more comparable to your gene of interest than are HK genes like GAPDH 
which are expressed at extremely high levels in the tissues I've looked 
at.   High expression of the HK gene as a control in an RT-PCR assay is 
often overlooked but I think can be of real concern when the cycle number 
is high enough that the PCR is no longer quantitative (out of linear 
range of amplification given the starting number of templates).  Perhaps 
this discussion should include information on what people are doing to 
ensure that their RT-PCR assay is quantitative.  My bet is that people 
aren't doing enough but aren't being required to for publication.  I 
would like to hear what others on the net think.

Darin (darin at gene.med.umn.edu)





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