Semi-quantitative RT-PCR

David Leavesley dleaves at
Mon Dec 14 00:43:53 EST 1998

Being a 'new kid on the block' as far as PCR is concerned, I have read with
interest the Quantitative PCR thread.
I still have serious reservations re its validity, in spite of frequent
publications in peer reviewed journals.

My problem is that I need a quantitative PCR protocol for a lab-based
clinical project examining the effect of LPS and cytokines (drugs) on our
(inducible) target gene during inflammatory responses. I have both biopsy
and primary, cultured material, and thus far, expression (yes or no)
fortunately correlates. However my clinical colleagues would like me to
demonstrate dose-dependant 'cause and effect' by Quantitative PCR. Before
you ask, NO, they have no idea what is involved! They have seen it
published and thus it 'must be right'!

To date I am post-staining with Molecular Probes' SYBR-Gold. It looks good
but how can I validate the results? I have also stained with EtBr, which
regreatably gave a subtly different result.

Any advice / directions / references (other than Raeymaekers, Becker-Andre
& Hahlbrock, or Alard) appreciated.

Affiliate Senior Lecturer, Dept. Medicine, University of Adelaide
Renal Unit Research Laboratory, Hanson Centre for Cancer Research
Dept. Renal Medicine, Royal Adelaide Hospital
North Terrace	ADELAIDE 5000	South Australia
ph:+61-8-8222 5203 (Sec), +61-8-8222 4990 (Lab), fx:+61-8-8222 5907

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