gino.miele gino.miele at bbsrc.ac.uk
Mon Dec 1 07:54:37 EST 1997

Lets set the record straight about DDRT-PCR!  This technique recieves an amazing amount of bad 
press, which I find utterly astonishing given that has major advantages over other methods for 
identifying differentially expressed genes.  It is an extremely powerful tool.  I work in a 
research group of 6 scientists and we all routinely use DDRT-PCR for a variety of different 
purposes, using RNA extracted from a wide variety of material, ranging from cultured cells, 
specific tissues to whole embryos.  We have been using the technique for the last couple of years 
with a great deal of success.  My particular project is concerned with hunting for brain 
transcripts up- or down-regulated in developing mouse brain as a direct result of the targeted 
deletion of a specific gene.  This is designed to give us clues as to the function of the encoded 
protein (the null has no phenotype).  For this project I am using whole mouse brains from a 
variety of wt/null developmental timepoints, which DDRT-PCR allows me to display side-by-side.  
No other method can do this!  I reckon that I've screened somewhere in the region of 70% of all 
transcripts expressed in the developing brain, almost all of which show no difference in 
expression levels between wt and null brain.  However, the technique has allowed me to identify 
various differentially expressed transcripts, in addition to identifying the one that was 
targeted.  These all confirm by northern analysis, although two require the use of Poly(A) RNA to 
give a signal, which proves to me that the technique is sensitive and displays even rare 
transcripts.  The technique has allowed me to identify genes downstream of the targeted gene and 
genes upregulated as a result of its ablation.  In short, our whole lab' has no problem 
whatsoever with the technique and we firmly believe (and have proof) that it is both sensitive 
and highly reproducible.  We find it works perfectly well on total RNA extracted from cultured 
cells, specific tissues eg. brain, or even whole embryos.  The DD gels themselves are undoubtedly 
an accurate picture of gene expression at that given time.  
I would suggest the following to anyone using it or planning to use it:-

1. Total RNA is fine.  Rare transcripts are still displayed.  There is NO bias 
towards transcripts of high abundance.  RNA quality and accurate quantitation is 
absolutely critical for success.  Quantitate RNA for each sample each time cDNA is 
being made.  We use Pharmacias'First Strand cDNA synthesis kit.

2. The original Liang and Pardee protocol is the one we use and find it works 
beautifully.  The use of random 10-mers is essential to give maximal coverage.  We 
have tried a variety of the kits on the market and to tell the truth, none of them 
are as good as following the Science protocol with your own primers etc.  Some of 
the kits on the go at the moment use longer random primers with restriction sites 
incorporated.  All the random primers thus have a common sequence and I don't 
believe for a second that this doesn't bias the display and reduce coverage, the 
'randomness' of which is the essence of the technique.

3. Pair-wise comparison of material is NOT satisfactory in our experience.  By far 
the best system to use is a timecourse of some sort.  For example, using developing 
brain - this results in the majority of bands appearing identical at all stages in 
all lanes - however, developmentally regulated genes will change in intensity.  Both 
these scanarios act as controls.

4. The major reported problem for DDRT-PCR comes at the stage when a candidate has 
been identified, ie. the high false positive rate.  Isolation of the DNA from the DD 
gel, cloning and sequencing frequently reveals the presence of more than on DNA 
species.  This is because a single band on a display may frequently contain 
co-migrating DNAs of identical size.  Reamplification of this mixture of DNAs for 
standard 40 cycles will result in all species being amplified to similar 
levels (CoT effect).  Most researchers opt for T/A cloning strategies for cloning DD 
PCR products, and it is well known known that the effiency of this is very sequence 
dependent.  The net effect of this is that co-migrating DNAs may clone with far 
greater efficiency than the DNA of interest (we have many, many examples of this), 
which may not even clone at all!  We beleive that this accounts for a very 
significant proportion of the observed false positive rate for DDRT-PCR.  Since 
incorporating measures to circumvent reamplification of comigrating DNAs we have 
reconfirmed by northern, RPA, ISH a lot of the candidates identified in different 
systems as being differentially expressed.  The reason I say a lot and not all is 
because we have yet to investigate a few more.

My advice is not to dump the technique.  Yes it is technically challenging, even 
although it looks simple on paper.  But with a bit of perseverance it works wonders, 
no matter what material you want to work with.  In no way is subractive 
hybridisation etc by any means a substitute for this technique - you can't analyse 
multiple samples, it is much more time-consuming, you need much more starting 
material and don't have anywhere near the same chance of identifying rare 
differentially expressed transcripts.

Gino Miele

E-MAIL:    gino.miele at bbsrc.ac.uk  

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