RT-PCR and Template Concentration

mbarnhart at my-dejanews.com mbarnhart at my-dejanews.com
Thu Mar 18 18:11:51 EST 1999


Duncan et al.

I have accounted for all of the problems below.  The same RNAs give signal
when spiked into a total RNA prep before RT but not when subjected to RT
without additional RNA.  Also, it's two tube using dT15 as primer in the RT
step.

Thanks,
Michael


In article <u8jjeEASTM82Ewns at genesys.demon.co.uk>,
  "Dr. Duncan Clark" <Duncan at genesys.demon.co.uk> wrote:
> In article <7cp5e9$3hv$1 at nnrp1.dejanews.com>, mbarnhart at my-dejanews.com
> writes
> >Does the amount of template RNA affect the efficiency of MMLV RT reactions?
> >When I have a very small amount of RNA (approx. 10-50 pg of cloned RNA
> >template), I get no signal in subsequent PCR reactions while I see plenty of
> >signal when I use a total RNA preparation containing approximately the same
> >number of molecules of the target RNA as in the reaction above.  The problem
> >might also be in the PCR reaction.  Any help would be appreciated.
>
> From the way you say it you appear to be generating an RNA transcript
> from a plasmid containing your cloned RNA. How are you doing your RT-
> PCR? Two tube or one. If two are you using one primer in the RT then two
> in the PCR and is the single primer in the RT correct for the way you
> have cloned your RNA and the subsequent transcript. What I'm getting at
> is have you cloned your RNA the wrong way around so the RT primer will
> not anneal?
>
> Again assuming your are using an RNA transcript, you must have used
> RNase free DNase to destroy the plasmid. Can you be sure that your RNA
> survived?
>
> Duncan
>
> --
> I've learned that no matter how thin you slice it, there are always two sides.
>
> Duncan Clark
> DNAmp Ltd.
> Tel: +44(0)1252376288
> FAX: +44(0)8701640382
> http://www.dnamp.com
> http://www.genesys.demon.co.uk
>

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