Good HKG for quantitative PCR

ERIC CRAWFORD crawford at comb1.comb.umd.edu
Tue Feb 23 08:23:00 EST 1993


In article <1mblq1$hc3 at gazette.bcm.tmc.edu>, 
Jorges at bcm.tmc.edu (Jorge L. Sepulveda) writes...

>In article <2B870CD4 at adminbldg.lan1.umanitoba.ca> 
>GIETZ at bldghsc.lan1.umanitoba.ca writes:
>> Hello Fellow Netters,
>>     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?

> 
>If you pick up primers from different exons (i.e.) separated by introns in 
>the genomic DNA, there should be no problem with unexpressed genes.
> 
>Cheers,
> 
> 
>Jorge L. Sepulveda, 
> 
>Dept. of Pathology,                        Email: Jorges at bcm.tmc.edu
>Baylor College of Medicine                 Telephone: 713-798-7330
>One Baylor Plaza, Houston, TX 77030        Telefax: 713-798-5838

Hi all,

Jorge's comments are correct IF there is no genomic DNA "contaminating"
the RNA to be RTd into ss-cDNA. I have used primers for two different 
exons to amplify RTd RNA and obtained two products:

1) a large product whose size is predicted if the size of the intron
   seperating the exons is included 

2) the smaller, predicted product presumably from the RTd mRNA

I can isolate DNA-free RNA (relatively) by using a gentle NP-40
lysis of my cells, pelleting the nuclei, DNase treatment of the
resulting "cytoplasmic" supernatant and EtOH ppt of the total
"cytoplasmic" RNA. Using this RNA prep I (usually =8-}) don't
have a problem with DNA contaminoma.

I have DNase treated RNA isolated using the guanidine thiocyanate/
acid phenol (complete lysis of the whole cell including nucleus)
method with mixed results.

Good luck,
	-Eric 

    Eric Crawford	      |      crawford at comb1.comb.umd.edu
    University of Maryland    |         Voice: (410) 706-5664
    School of Medicine	      |           FAX: (410) 706-8162
    10 South Pine Street      |  "standing on the shoulders of giants
    Baltimore, MD 21201 USA   |          makes me tall"   =(8-} 



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