RT-PCR PROBLEMS

Pamela Norton pnorton at lac.jci.tju.edu
Wed Jan 17 13:58:40 EST 1996



> >>I have been trying to amplify messages using RT-PCR, but I don't get 
> any
> >>bands after the PCR.  I failed to see bands even with primers from
> >>Clontech designed to amplify G3PDH.  The protocol that came with the
> >>primers recommended that I use oligo-dT or random primers during the 
> RT
> >>step, but I just used the 3' PCR primer.  Has anyone out there have
> >>success with RT-PCR without using oligo-dT or random priming?  
> >
> >it does not work. 
> >I (and others) tried it before.
> >The reason is very simple- your PCR primer is just too long
> >and anneals everywhere at the tempr of rev transcription.
> >There are some hints:
> >
> >1. Try rev transcription at 50 C- SuperScript RT
> >   survives at 50C
> >2. Reduce the conc of the primer during rev transcription
> >3. If 1 and 2 do not work you have to use oligodT or
> >   (as we did) design a short specific antisense primer
> >   with Tm around 37-40 C.
> >
> >Andrei
> 
> That's right.
> but if you don't want to purchase superscript or other thermosatble 
> rev transcriptase and still like to use specific primers for the RT 
> step, you might use you original 3' primer for RT and use a second 
> NESTED 3'  primer (i.e. upstream of your RT primer) for your PCR. this 
> also circumvents the problem of mispriming to non-specific sites.
> 
> Sui Huang

(I edited some extra lines out of the above exchange to shorten it but
have not changed the attributions. Unfortunately, the original posters
name has gotten lost somewhere along the way, apologies to him/her.)

Greetings,

   I'm confused by this thread. So what if the gene-specific primer
misprimes at other places? How is this different from random priming,
where the specificity lies in the PCR reaction? The only problem that I
can see is if an insufficient amount of primer was used in the first
strand reaction. 


   For what it's worth, I once did an experiment comparing the use of
random primers, oligo dT and a specific 3' primer in the RT reaction,
followed by PCR with the same 3' primer and an appropriate 5' primer. The
oligo dT was the least effective, probably because the region being
amplified was a couple of kb upstream of the polyA tail. The other two
methods both worked fine. Anyone else like to share their experiences?


   At this point, I generally use random primers, but I do not see why the
original poster's experiment should not have worked. I suspect that the
problem lies elsewhere.

      Pam Norton

-- 
Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



More information about the Methods mailing list