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Semi-quantitative RT-PCR

Samuel Gray samuel.gray at nottinghNOSPam.ac.uk
Tue Jan 18 11:16:47 EST 2000


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.







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