Good alternative for Quantitative PCR

frist at ccu.umanitoba.ca frist at ccu.umanitoba.ca
Thu Feb 25 15:14:49 EST 1993


In article <9302221844.AA04759 at phobos.med.pitt.edu> bsh at MED.PITT.EDU (Basavaraju Shankarappa) writes:
>>     I am posting this question for a friend
>> who is not hard wired to the net yet.  Does anyone
>> know of a gene or genes (of house keeping variety) other than actin or
>> G3PD that can be used for standardizing quantitative PCR from
>> RTed mRNA extracted from mammalian tissue samples?  The problem
>> with these two genes, I gather, are the psuedo genes that amp
>> from the DNA also found the nucleic acid prep. There has been no luck 
>> so far with with DNAseI digestion, followed by phenol extraction and 
>> precipitation
>> (using glycogen as a carrier).  A simple fix is to use a house keeping gene
>> that has no psuedo genes.  Are there any currently used for this purpose?
>> Thanks in advance!!!!
>> ________________________________________
>> Dan Gietz
>> GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
>> _________________________________________
>
>	I have been reading recently about another good method for the 
>quantitative PCR.  Here, you make a construct of the gene you are trying
>to quantitate, which has an appreciable amount of deletion inthe construct.
>Now you can add this modified construct to the template in highly defined
>proportions such that this template also uses the same primes as the one
>you are trying to quantitate but will yield a different sized band on an
>agarose gel.  A photometric or other method of comparison will give you
>a much better and consistent and reliable method for quantitating the 
>unknown product.  There were some papers in recent issues of Science, but
>I cannot recall which one.  

White, M.J. etl al. (1992) Expression of the chlorophyll a/b-protein
multigene family in pea (Pisum sativum L.) Planta 188:190-198.

describes a simple PCR assay using chemiluminescent detection of RT-PCR
products with an internal standard, as you describe here. Deletion
standards are made directly by PCR, eliminating cloning steps.
Chemiluminescence is quantitated by densitomitry of X-ray film, as you 
would do for any autoradiogram. 

>	I think this method will replace the methods that use housekeeping
>genes.
>	Raj Shankarappa
>bsh at med.pitt.edu

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