help with semiQ RT-PCR

[Patrick HJ Falckh]

Proteins are not always possible to isolate at sufficient purity or 
completely active thereby giving a false representation of the protein 
produced.  If no controls are present to ensure optimal PCR conditions 
or variations between PCR runs, then people shouldn't use the words 
quantitate/quantitative at all!  mRNA levels are only sufficient to 
measure chronic changes, in some cases, as the level is transitory and 
upon the removal of the stimulus the mRNA levels return to normal; 
such is the case with induction.

A change is only a change if other criteria stay constant; ie a 
standard.  As mentioned in other replies at earlier postings, the 
difficulty arises on choosing THE RIGHT standard, and in most cases 
more than one standard should be considered.

There is nothing wrong in denoting that a tissue expresses a gene, 
provided you have accounted for the possibility of DNA contamination, 
and are aware if the gene being expressed has any introns or not; eg 
muscarinic receptors have no introns, so a DNase step is imperative 
before being able to denote expression or a change in expression.

Just my $0.02 worth.  I know that it's still "publish or perish" but 
I'm starting to get annoyed with papers that have been published that 
haven't taken into account any controls and when trying to replicate 
the experiment nothing works!  On trying to use a method in a recent 
'Proceedings of the National Academy of Science, USA' I had to resort 
to contacting the author directly, after several unsuccessful attempts 
to get the experiment to work, before finding out that the papers 
results were on a N=1.

There is enough bad press against scientist without us giving credence 
to those statements with sloppy work.

Regards
-- 
                                                                    
   Patrick HJ Falckh PhD                                 #     #     
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