quantitative RT-PCR

Chris Kafer ckafer at iastate.edu
Tue Nov 9 23:40:59 EST 1999


Hi.  I'm currently using 18s ribosomal as the internal standard.  I'm
using a homegrown "competimer" that consists of an identical sequence
to both 18s primers but w/ a 3' C3 spacer to inhibit elongation during
the PCR.  This will hopefully solve the problems mentioned below.

See the Ambion web site for some good info on qRT-PCR.



On Wed, 10 Nov 1999 08:56:58 +0800, Alec Redwood
<aredwood at cyllene.uwa.edu.au> wrote:


>I don't know much about internal standard for plants, however I suggest
>that you find an internal control that is present in approx the same
>concentration as your test otherwise you will run into all sorts of
>problems (competition for reagents etc.).  Alternatively if you have to
>use an internal control that is greater in conc. than your test then you
>could try adding control after 10-20 cycles of your test alone (primer
>dropping).  See ya have fun
>





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