Samuel Gray wrote in message <8623il$k9i$1 at oyez.ccc.nottingham.ac.uk>...
>Dear researchers,
>>Can anyone offer any suggestions as to how to increase the sensitivity of a
>RT-PCR system? We are trying to perform simple semi-quantitative RT-PCR on
>muscle biopsy samples, comparing a PKC isoform mRNA with GAPDH mRNA levels,
>but however I adjust cycles, template concentrations and conditions I
cannot
>detect any PCR products within the linear amplification range - only at
>plateau.
>>The limitation is that we cannot use isotopes at all in our (small)
>department so I am having to detect our PCR products by ethidium bromide
>fluorescence on a Syngene ccd gel-doc system and the sensitivity is just
too
>low I think.
>>Any suggestions would be gratefully received!
>>Yours,
>>Samuel Gray
>Division of Vascular Medicine,
>University of Nottingham,
>Derbyshire Royal Infirmary,
>Derby,
>DE1 2QY.
>>Samuel,
You may want to try some alternatives to Ethidium. Molecular Probes
(http://www.probes.com) sells a large number of DNA-binding dyes which are
reported to help visualize small amounts on a gel. If you have access to a
fluorometer, you could try one of the DNA specific dyes available from
molecular probes when you do not see anything on a gel.
Alternatively, if you have a plate reader you could try digoxigenin
incorporation and visualization using an anti-dig antibody. This should
give you a little greater sensitivity.
Hope this helps!!!
Jeff Fairman
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