RT-PCR

[Osama]

Dear All,

I have been increasingly facing problems with RT-PCR. It worked after 
three trials at the very begining. Now, I am trying it for more than 4 
times with no success. I have noticed that if I change the quantity of 
the first strand cDNA into the PCR reaction that the intensity of the 
band changes. Amazingly, the less the quatity of the cDNA the stronger 
the band up to a certain threshold. 

Is the first strand cDNA quantity so crucial in the PCR reaction or am I 
doing something wrong?  How do you know that the result of PCR is optimal 
and really representative of mRNA monitored and is not due to imperfect 
PCR reaction? Is using positive control for a gene which is switched on 
all the time enough for quatitification of RNA? 

I am really lost in a PCR which does not work and the bias in results....?

Any comments...


Regards,

Osama

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Osama A. Al-Assar                  .     ||\       * *           *.
N. Ireland, UK                     .     ||'\       * *         *.
EDQ424 at dsets.ulst.ac.uk            .    / |''\       * *       *.
Research Student                   .    \ |''/        * *     *.
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                                   .   /..|/            * * .
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