RT-PCR vs. RNase protection assay

Pamela Norton pnorton at lac.jci.tju.edu
Tue Jun 18 17:58:16 EST 1996


In article <Pine.3.89.9606141211.A13834-0100000 at darwin>,
shelley at DARWIN.SFBR.ORG (Shelley Cole) wrote:

> Several contributors to this newsgroup have stated that ribonuclease
> protection assays (RPAs) are more sensitive and therefore probably
> more reproducable/reliable than using quantitative RT-PCR as an assay
> for gene expression.  I have been trying to convince a colleague of this,
> and the usual theoretical arguments are not working. He has heard
> that RT-PCR is more sensitive and a lot cheaper than RPAs.
                      ^^^^^^^^^^^^^^^^^^^^^^^^^

RT_PCR is more sensitive than RPA, making it valuable when RNA quantities
are limiting; you didn't mention whether this was a factor in your case.
However, I strongly suspect that RT-PCR is more expensive than RPA, due to
the high cost of the enzymes. The most significant expense for RPA is the
radiolabelled nucleotide, but if you can make more than one probe with a
single batch, the economics are not too bad.

>   Could any of you direct me to some peer-reviewed publications that
> discuss comparison of these methods?  I have some review articles on
> each method, but none that deal directly with comparison.

Please share this information if you obtain it.

>   Also, I have already developed a RPA for one of our genes of interest.
> I have never done quantitative RT-PCR, so can anyone give me a realistic
> estimate of the amount/time of work that would go into developing an
> accurate quantitative RT-PCR assay.

We were trying to perform quantitation of genomic DNA by PCR and gave up,
it required lots of trials to establish that one was in the linear
amplification range, and was cumbersome. Consider that we did not have the
extra variable of needing to perform the RT step. 

Personally, I feel that it is much easier to obtain quantitative data by
RPA, the main limitation is your ability to accurately quantitate the
amount of RNA present in your samples (this limitation applies to all
methods, however. 

Hope this helps,

   Pam Norton

-- 
Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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