Quant. RT-PCR: continued

suihuang at usa1.com suihuang at usa1.com
Thu Aug 31 22:22:22 EST 1995


There was quite a polemic discussion about the validity and 
reliability of quantitative RT-PCR. And it seems that there 've 
been some (terminological) confusion. 

Don't we have to distinguish between these two different 
settings:

A) ABSOLUTE quantitation:(How many copies of messengers are 
present?)
For this kind of questions people have been using COMPETITVE 
pcr: Two templates, the unknown and the standart, compete for 
the SAME set of primers, but give rise to distinguishable 
amplicons (eg by size or retriction site), which MUST amplify 
at the same rate.

B) RELATIVE (semi) quantitation: (How does the expression of a 
given gene changes in different situations, eg. stimulated vs 
non-stimulated tissue). This kind of question has been adressed 
by MULTIPLEX PCR: Two different genes (RT products) are 
amplified with two DIFFERENT primer pairs. The amplification 
efficiency might be different for those two amplicons, but the 
ratio of these efficiencies is expected to be constant in 
different RNA preparations. If the PCR is stopped before 
plateau is riched, the ratio of pcr products should reflect the 
raton of the input number of the templates.Thus this is 
COMPARATIVE rather than COMPETITVE. One gene can be the 
"internal standart", eg a hose-keeping gene (indeed, as 
previously mentioned by Tracy and Alexandre, GAPDH is not 
appropriate: it is also growth-dependent), while the other gene 
is which one is interested in measuring changes in messenger 
stady-state levels. 


Most of the dispute about quantitative PCR, i think, refers to 
the COMPETITIVE type, since the choice of good COMPETITORS is 
often a cetral issue. 
There are, however, several examples of RELATITIVE quantitation 
published. 
Could anyone tell me whether there have been a simialr 
discussion on this type of RELATIVE quantification ??
Any comments, suggestions etc appreciated.

Sui Huang




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