Does RNA - PCR ??

Richard R. Hardy hardy at mighty.fccc.edu
Mon Jul 17 09:05:45 EST 1995


In article <DBusA7.Lv5 at info.swan.ac.uk>, BABAKER at Swansea.ac.uk wrote:

>Does RNA PCR??
>
>I am doing RT-PCR and get positive results even when just PCRing RNA
>using cDNA primers and Taq DNA Polymerase, with no cDNA or genomic DNA 
>anywhere close by!!
>
>Is it possible that the gene sequence of the mRNA which would only
>contain 1 or 2 U's instead of the T's in the primers sequence would be
>amplifiable by the cDNA primers? As annealing would probably jump the
>U's in the mRNA for that gene and then continue in the PCR to give 
>the correct sized band?
>
>I dont think that DNA is contaminating my RNA prep as I use the
>Acid-Guanidinium thiocyanate method of isolation, which should 
>erradicate the DNA.
>
We have no problem amplifying trace DNA contamination from this type of
"RNA" prep - treat your samples with good-quality DNAase.  Or design
primers that span exons so that the DNA and RNA are different sizes... 
Good luck!

-- 
Richard R. Hardy
Member, Institute for Cancer Research
Fox Chase Cancer Center, Philadelphia, PA
(215) 728-2463 - voice
(215) 728-2412 - FAX



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