RNA + PCR

Gary Paterson g.paterson at bio.gla.ac.uk
Wed Apr 29 06:17:07 EST 1998


I have a problem at the moment.  I am interested in doing quantitative
RT-PCR on a number of RNA targets.  When I perform PCR on DNase1 treated
RNA, I get products of the correct size and when I perform PCR on RNA
not treated with DNase1, the gel has more contaminating bands with the
most intense ones migrating slower since I presume I am amplifying the
genomic copies which contain introns.  I can only assume that the reason
the band intensities are the same ( albeit different in size) is because
the level of expression is so low that it resembles the gene copy
number.  If this is true, I'd expect  2 equally strong bands from RNA
contaminated with DNA, with one corresponding to the RNA template and
one corresponding to the genomic template, however, for some reason the
PCR reaction favours the larger genomic product!  Any clues why?
Another related problem is that when I use Dnase treated RNA and perform
the first strand cDNA synthesis reaction without using reverse
transcriptase, any bands detected on a PCR gel I would presume would
result from residual amounts of undegraded DNA.  Yes I get these bands,
however, although the band intensities are less, the bands migrate the
same as the ones from Dnase treated RNA and not from the DNA
contaminated samples?  Does Taq pol have residual RT activity?   I'm
using Platinum Taq from Life Technologies.

Any help would be most appreciated

Gary




More information about the Methods mailing list