quantitative RT-PCR

[Isaac Kim]

Organization: Northwestern University
Reply-To: ikekim at merle.acns.nwu.edu
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In article <aquilla.1160010482G at sadye.emba.uvm.edu>, 
aquilla at salus.med.uvm.edu (Tracy Aquilla) says:
>
>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.)
>
>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


To make RT-PCR quantitative, you must use primer sequences that are 
identical to your target because different primers have different 
efficiencies of amplification. To do this, you can clone the RT-PCR 
product and either delete or insert a fragment into the PCR product. Now 
this 'new' product has same sequence for the primers as the target but 
can be distinguished by size on an agarose gel electorphoresis. PCR using 
this 'new' product is truly competitive in that the amplification 
efficiency is equal to the target. By knowing the amount of the 'new' 
competitor that you have added to your RT-PCR runs, you can quantitate 
the amount of the target present originally in the RNA prep. Of course, 
you have to deal with the efficiency of the reverse transcription if you 
want to know the "actual" amount of the target. To do this, you can do in 
vitro transcription of the competitor and add a set amount of competitor 
RNA to the RT-PCR. I will be more than happy to give additional 
references if anyone wants them.

Now, I must make some comments about the comparison between Northern blot 
analysis and competitive quantitative RT-PCR. In my experience, 
competitve quantitative RT-PCR has been just as accurate as Northern blot 
analysis in measuring the relative changes in the level of mRNA.(Yes, I 
have actually done the comparison of the two before using competitive 
quantitative RT-PCR.) Moreover, there is a question as to what the 
standard for equal loading is in Northern blot analysis. As most of my 
experiments use hormone treatment, I am not sure that the level of any 
mRNA encoding housekeeping protein is constant. (I have also tried 
beta-actin.) Just as my GAPDH mRNA seems to be changing in response to 
hormone treatment (as it was pointed out by Tracy), can anyone assure me 
that the level of any other protein's mRNA is not changing in response to 
hormone treatment? In fact, it is hard for me to imagine that there is a 
housekeeping protein whose mRNA level does not change with hormone 
treatment as my cells undergo proliferation with hormone addition. Then 
conversely, if there is a protein (used as standard) whose mRNA level 
seems to be constant via Northern blot analysis in all hormone treatment, 
can you be sure that you did not under load some samples and as a result 
you see "equal intensity" of the band? Is there turly a mRNA specie whose 
level "does not change" in response to hormone treatment? If there is 
such an ideal mRNA specie, please let me know. I will be glad to try them 
in my works.

Under these conditions, I feel that competitive quantitative RT-PCR can 
be more accurate than Northern blot analysis because with competitive 
quantitative RT-PCR, you specifically add same amount of 
standard/competitor for all samples.

Isaac.