'semiquantitative' RT-PCR

Steven Sullivan sullivan at gwis2.circ.gwu.edu
Tue Jun 30 00:30:21 EST 1998


I see lots of papers with 'semi' or comparative RT-PCR data in the
developmental biology journals -- e.g. one amplicon compared in different
treatments or stages, with amplification of a 'housekeeper' gene as a
normalizing control.  I'm still a bit unclear as to how teh authors
determine 'linearity' in these experiments though.  For a given primer
set, must one test 1) a range of cDNA input amounts, or 2) a range of
cycle numbers?  And is this done for *each* different RNA sample (e.g. 
injected versus uninjected Xenopus animal caps) or can one jsut use any
tissue in which the target RNA is expressed (e.g. a whole Xenopus embryo) 
*once* and never have to do it again for that set?  People I've spoken
with seem to have different ideas over which combination of these
approaches constitutes 'rigor'.  I've already encountered the phenomenon
of being able to RT-PCR up a supposedly 'unexpressed' mRNA simply by doing
more than the published number of cycles. 

None of the PCR protocol books I've seen discuss this in detail, but if
anyone can point me at a good reference please do. 




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