eGFP transcript quantification using LightCycler RT-PCR
king-ferdi at gmx.net
Mon Feb 6 04:36:12 EST 2006
I am trying to quantify the eGFP transcription in eukaryotic cells (HeLa
and CHO) employing a LightCycler 1.5 RT-PCR system. The primers were
designed using the primer3 program.
Whole RNA the transcript doesnt contain a poly(A) tail - is isolated
using the High Pure RNA Isolation Kit Roche. cDNA is gained using one
specific primer by incubating with RevertAid H Minus M-MuLV RT
Fermentas at 55°C for 1.5h. The RT-PCR is performed in a LightCycler 1.5
RT-PCR system using the LightCycler FastStart DNA MasterPLUS SYBR Green
I Kit Roche.
Even after fine tuning of the PCR program (T(Annealing); t(Annealing);
t(elongation); [primer]) I have a high background using RNA isolated
from cells not expressing eGFP. This background is no smear, but
Does anybody has any suggestions how to get rid of the background or if
there is any program on the net to virtually test primers on total RNA
(hamster and human cells).
I searched all RT-PCR primer databases I know for tested eGFP primers,
but found none. So if anybody has the sequences of working RT-PCR
primers used in transcript-quantification, I would be really happy if
you could post them...
Thanks in advance,
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