eGFP transcript quantification using LightCycler RT-PCR

Michael Sullivan 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?

Mike

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 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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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)




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