IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

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
>detect any PCR products within the linear amplification range - only at
>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
>low I think.
>Any suggestions would be gratefully received!
>Samuel Gray
>Division of Vascular Medicine,
>University of Nottingham,
>Derbyshire Royal Infirmary,
>DE1 2QY.

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


visit:  http://www.thelabrat.com  - By Scientists... For Scientists.

More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net