eGFP transcript quantification using LightCycler RT-PCR

king-ferdi 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 doesn’t 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 
specific bands.

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,

Fernando



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