Re: RTPCR from paraffin sections
In a message dated 5/5/2003 9:49:09 AM Eastern Standard Time, Nancy.Walker@sanofi-synthelabo.com writes:
> Using the PALM microdissection system, have no sucess in amplifying mRNA
> from paraffin (PFA fixed) sections, while with frozen
> sections RTPCR with
> single cells is possible. Anybody know a trick?
I have a feeling the problem lies in the lability of the RNA and its sensitivity to denaturation/degradation. There may not be any intact RNA left in the paraffin-embedded tissue.
As I understand it, proteins are the hardiest; if you microdissect a large number of paraffin-embedded specimens and combine them, you might be able to get enough protein to work with.
DNA is more fragile than protein, and you could possibly microdissect and extract enough DNA to see something, but I'm not sure I would trust the results too much.
RNA is the most fragile of all; even under the best of circumstances (frozen sections) you would need to work as quickly as possible to prepare, freeze, section and microdissect the specimens. Autolysis and degradation begin immediately, so speed is of the essence.
I hope this helps. There are probably others on the list who do this with more regularity than I do, and they can probably shed more light on the problems you're encountering.
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Desert Southwest Region, USA
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