Problems with RT-PCR

rover at livnet.com rover at livnet.com
Wed Jan 10 15:40:26 EST 1996


karbach at wbzx06.biozentrum.uni-wuerzburg.de (Doris Karbach) wrote:

>Dear Netters,
>I am doing quite a lot of RT-PCR and I was sure to convince all initial 
>problems. Now I want to detect the mRNA of one protein, which I could detect 
>rather good in Western Blots. I am sure that my RNA is not degraded and 
>Primers fit very well. I have raised the concentration of MgCl2 and it went 
>only slightly better. Has anyone an idea what to make better
>Thanks in advance Doris

My first question is whether your RNA is being expressed at a high
enough level to be detected ... is the protein continously present?
,is it in response to a stimuli?, is it a paracrine or exocrine, is
this from tissueor cultured cells?. Since we don't know what your
trying to detect. It is quite possible that your RNA expression has
already declined post translation of the protein, and that the half
life of the protein is long enought to enable detection, or that the
the translation of the protein is occuring in another cell type or
tissue. The detection of the protein does not necessarily mean that
the RNA is currently being expressed when and where you are looking.
Without more information I cannot give anymore advice.

- do you have a control for your RT-PCR...B actin, etc? To check the
method?


Rover The Mad Molecular Biologist
rover at livnet.com
barlow at picard.evms.edu




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