Quantitative RT-PCR

ELADER at AMBION.COM ELADER at AMBION.COM
Fri Jun 19 08:50:40 EST 1998


In article <6mb9vi$hva$1 at nnrp1.dejanews.com>,
  mbarnhart at my-dejanews.com wrote:
>
> Hi,
>
> We are developing competitive RT-PCR for three genes and plan to work with
> more in the future.  Since we typically only use about 20% of the RT
> reaction, I was wondering if we could use multiple competitors in a given
> reverse transcription so that one reaction could be used for multiple genes.
> For example:
>
>     RT rxn 1:  Sample RNA + 10 e10 copies Actin competitor + 10 e10 copies of
> myosin competitor

Hi yourself,

What you are suggesting is theoretically sound. What complicates matters is
the possible interaction of the two pairs of unrelated primers and the
complexity of titering both synthetic RNA's in the reactions. If what you are
suggesting is to use (example...) sample 1 to quantitate the actin (where the
actin bands are equivalent) and sample 4 to quantitate gene x, I bet it would
work.

BTW, actin is a really miserable control since most likely your primers are
picking up several actin transcripts such as gamma actin. Make your actin
primers int he 3' untranslated region. This is less related between gamma and
beta.

Eric

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