quantitative RT-PCR

Frank Chen yatsen at wam.umd.edu
Mon Aug 28 16:44:08 EST 1995


I cannot help to reply to message after I read Tracy's reply and share a 
story.  I agree with Tracy's concerns.

In last Fall, Hybraid hosted symposium in Bethasda in an attempt to 
improve the sell of their new slide-PCR machine specifically designed for 
RT-PCR insitu hybridization.  The outcome was amusing:

A lot of invited speakers along with audience critisized the "usefulness" 
of RT-PCR insitu, especially for quantitative purposes.  One PI in our 
department also went the meeting.  AFter talking to one of the speakers 
during the lunch, she immediately realized that her RT-PCR insitu results 
were artifacts!  She felt lucky that she only presented the data in a 
poster in a conference but not in a publication yet.  She returned the 
machine after she came back and decided to stick with "conventional" in 
situ!  And, we concelled our decision to buy one!

PS: I didn't keep the abstracts of the meeting.  Anyone interested in who 
the speakers (authoratives?) are should contact Hybraid.

Frank


On Mon, 28 Aug 1995, Tracy Aquilla wrote:

> In Article <41kmno$84 at news.acns.nwu.edu>, ikekim at merle.acns.nwu.edu. (Isaac
> Kim) wrote:
> >I have used quantitative RT-PCR for past publications. I have had no 
> >problems with the reviewers. The main problem with RT-PCR is that 
> >different primers have different efficiency of amplification. If you 
> >choose the conditions of the standard correctly, RT-PCR can be more 
> >accurate than Northern blot analysis, in my experience. Even more
> 
> This is an interesting topic for discussion. I'd like to know if you have
> data to support this claim, or is this merely speculation? Specifically, how
> did you (accurately) measure the samples to compare the accuracy of RT-PCR
> versus Northern analysis?
>  
> >interestingly, a recent manuscript that I submitted to a reputable 
> >journal, the reviewers' response was that I should use quantitative 
> >RT-PCR over that of Northern blot analysis for all my studies as the 
> >control for Northern, GAPDH, showed variations in the level. I would like 
> >to direct you to two articles regarding this matter. 
> >Siebert PD et al. 1992. Competitive PCR. Nature 359:557-558
> >Forster E. 1994. Rapid generation of internal standards for competitive 
> >PCR by low-stringency primer annealing.
> 
> (I think you forgot to include the actual citation here.  WHICH JOURNAL?)
> 
> So, are you (and/or these reviewers) saying the Northern appears to be less
> accurate because it showed variation in GAPDH levels between samples? This
> is an invalid conclusion. How do know there REALLY isn't variation in the
> levels of GAPDH? In other words, maybe this is an indication that the
> Northern is actually MORE accurate than RT-PCR.
> 
> Furthermore, GAPDH is actually a pretty lousy internal standard. GAPDH
> levels vary significantly between individuals and with developmental stage,
> as well as in various tissues and with numerous experimental treatments.
> Furthermore, many different GAPDH-related sequences have been identified and
> there appear to be approximately 300 copies of these in the rat genome and
> 100 copies in the human genome. To make matters even worse, some of these
> pseudogenes are actually transcribed and fully processed (NAR 13:7,
> 2485-2502). This does not make for an ideal internal control! I'd say the
> real problem is with your standard, and not necessarily the method of analysis.
>     Tracy
> 
> 



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