RT-PCR PROBLEMS

Patrick HJ Falckh p.falckh at rmit.edu.au
Sun Jan 7 19:41:44 EST 1996


Andrei Popov <ANDREI.POPOV at bbsrc.ac.uk> wrote:
>Hello all,
>>
>>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?
>
>Hello there,
>
>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.
>
>
>best wishes
>
>Andrei
>

Andrei,
   I hate to be contrary but the method DOES work but there are inherent problems with it. I've got muscarinic receptor expression (G-protein coupled receptors) to function in a single reaction mix that will run both the RT and PCR, and in the same Mg conc.
   One of the problems is that the  DNA polymerase (I use Taq) has to be in excess of the RT enzyme.  My method is an extention of work published by Sellner LN et al, Nucl Acid Res; 20(7):1487-1490; 1992 where he showed that if the reverse transcriptase was 0.5U then the Taq conc. should be 2.oU to amplify from 20fg of viral RNA.
   As an initial trial try the following :

     Final Conc         Reagent
     ----------         -------
      1.0 mM            of each dNTP
      6.4 mM            MgCL2
      1.0 X             PCR buffer (std mix excluding Mg)
      0.225 U           reverse transcriptase (I used Promega AMV)
      1.0 U             DNA polymerase (I used Promega Taq)
      10 U              RNasin
      2.7 pmol          of each primer
     ======================    The reaction was completed in a capillary thermal cycler in a final volume of 10ul, however, I can't see why it wouldn't work in larger volumes in a different cycler.  The RT portion of the procedure was carried out for 45 min at 42C then a 3 step PCR cycler for 30-35 cycles which were previosly optomised.

    This will only work with ONE set of primers at a time- additional primer pairs will increase product variability (eg I got 137 different bands using a muscarinic receptor primer-pair and a tranferrin receptor primer-pair)


   Hope this is useful to you

Regards

Patrick

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