RT-PCR

[ldm at nwu.edu]

In article <47dgrc$5ug at fu-berlin.de>, bohnemeier at ukbf.fu-berlin.de wrote:

> Hi you all!
> 
> I've certain problems with my RT-PCR. I want to amplify a part 
> of the GAPDH-cDNA sequence performing first a RT with total mRNA
> and later continuing with a PCR and specific GAPDH primers.
> The primers are designed to overspann several introns even 
> overlapping an intron (i.e. the 3' part begins in one exon whereas
> the 5' part ends in the following one), so I can differenciate 
> genomic DNA and cDNA. RT-PCR works fine. I get the amplicon that
> I expect for cDNA.
> The problem: even if I don't use reverse transcriptase (RT) in my
> assay, I get the same PCR-product as done with RT, expecting not 
> to have any PCR product. Faults like contamination or changing samples
> are excluded.
> So I supose a) the sequences published for GAPDH mRNA (cDNA) and
>                the corresponding genomic DNA are not correct
>                (there might be polymorphisms laking 4 introns?),
>             b) my Taq-Polymerase (GIBCO) has a slight RT-activity
>                producing few cDNA molecules during the first steps of
>                PCR.
> My question:
>    Has anyone encountered with similar problems? Are there any ideas
>    about this phenomenon (for me)?
> 
> Thanks in advance for your answers!

The bands in your no RT samples may be coming from GAPDH pseudo-genes
which are present in the DNA contaminating your RNA.