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Semi-quantitative RT-PCR

Jeff Fairman jfairman at die.spammer.die.com
Tue Jan 18 12:28:50 EST 2000


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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