Quantitative PCR question - Comparative quant PCR with 16S rRna as reference gene (Apr/22/2008 )

I am working on determining the differential expression of genes involved in iron oxidation vs sulfur oxidation in a bacterial isolate using RT-PCR similiar to the following study:

http://aem.asm.org/cgi/content/abstract/73/8/2491

However, I am confused on their study and some others I have read (i.e. http://mic.sgmjournals.org/cgi/content/abstract/153/1/102)

These studies are comparative quant RT-PCR on mRNA for expressed gene products compared to the 16S rRNA gene. The values in their results are n-fold expression levels normalized against the 16S rRNA. They are reporting 1000x and greater expression levels relative to 16S. How is this possible since rRNA is >99% of the total extracted RNA (they extracted total RNA by the way)? This seems impossible. Am I missing something? Does extracted rRNA not amplify with RT-PCR (cDNA synthesis). I have read these methods over and over and do not understand?? Any help would be appreciated in understanding their methods.

Thanks

-koze9-

QUOTE (koze9 @ Apr 22 2008, 10:28 PM)

to rephrase my question:

I understand that 99% of the RNA isolated with my total RNA isolation kit (qbio problu kit) is rRNA. I am going to quantitate mRNA for specific genes and compare to 16S expression. Do my 16S primers amplify only mRNA for 16S or also all the rRNA? Stupid question maybe but I am confused on some recent studies that indicate over 1000x relative expression to the 16S which should be impossible if rRNA is amplified??

for example:

http://aem.asm.org/cgi/content/abstract/73/8/2491

and

http://mic.sgmjournals.org/cgi/content/abstract/153/1/102

thank you

-koze9-

the 16s primers will work with the 16s rrna (that's what that primer is for, and the first article which you cited points it out, sort of).

-mdfenko-