eGFP transcript quantification using LightCycler RT-PCR
mlsulliv at wisc.edu
Mon Feb 6 14:24:37 EST 2006
Is the specific primer you are using to make cDNA different from the
primer pair you are using for the quantitation? If not, you might try
that to get better specificity.
Have you done a positive control of any sort, e.g. something you know
you should get a specific amplification product for?
On Feb 6, 2006, at 3:36 AM, king-ferdi wrote:
> 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 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,
> Methods mailing list
> Methods at net.bio.net
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)
More information about the Methods