Semi-quantitative RTPCR

[Olivier Gandrillon]

Hi

This is a highly controversial question. From close to ten years 
experience in semi-quantitative RT-PCR, I would say that any REPETITIVE 
difference from a higher than FIVE TIMES ratio (i.e. five times more (or 
less) signal in the treated versus untreated condition) should be 
considered significant. How did yu ensure that "the cycle number has 
been limited to ensure that amplification is still exponential"? This 
has to be done with EACH new pair of primers. The efficiency of the 
reaction is higly primer dependent. A lot of people (mostly old Northern 
practitionners) will disagree but I am absolutely sure that in careful 
hands, this is AT LEAST as reliable as a Northern blot (some of which 
have been made in conditions I would not trust). I guess the basis for 
confidence is : repeat ad nauseam, even starting from the very beginning 
(i.e. new cells). And don't trust two times differences (what does that 
mean in physiological  terms, anyway???). Then you're sure. Well, I am!

Hope this helps.

Olivier


Helen Haase wrote:
> 
> Hi!
> I would like to know more about validating my semiquantitative RTPCR
> system.  From the same RT sample I am doing two PCR reactions, one
> using primers for the message of interest and one for
> B2-microglobulin, which should act as a control.  The PCR reactions
> are done under the same conditions, at the same time.  I have
> established that the cycle number has been limited to ensure that
> amplification is still exponential.
> 
> Is it valid then to compare the intensity of staining of the ethidium
> bromide stained bands and express results relative to the B2M control?
> Any references on principles involved would be appreciated
> 
> Thank you
> Helen