CLONTECH PCR-Select

Dom Spinella dspinella at chugaibio.com
Wed Nov 19 11:51:23 EST 1997


James McKay writes:
> Has anybody had any experience in using Clontech's PCR-Select cDNA
> Subtraction kit? Specifically with regard to finding differences in
> oncogenes expressed in cancerous and normal tissue?
> 
> Thanks in advance,
> 
> James

James:

Yes, we have used PCR Select to look at cancers. The system does work in
that you will discover some differentially expressed genes (including a
few oncogenes) that seem to correlate with the tumor phenotype.  You
will also find a lot of mitochondrial genes, as well as a fair number of
"false positives" that do not pan out upon Northern Blot or RNAse
protection analysis for example.  If you're just interested in known
oncogenes, you're much better off screening for them directly by blots
(or better, micro-arrays) or even by quantitative PCR.

The fact of the matter is that there are literally hundreds (maybe even
thousands) of gene expression differences between "tumor" and "normal"
cells -- even of the same lineage. The discovery of such differntially
expressed genes is not the problem -- almost any technique you employ
(including just picking random cDNAs) will find a subset of them.  The
real issue is determining their relevance to the disease. First of all,
what constitutes a "tumor" and a "normal" tissue sample?  If its just a
hunk of tissue removed by a surgeon, there may be many different cell
types in the sample (e.g. parenchymal cells, epithelial cells, vascular
cells, blood cells, inflammatory cells, etc. etc., as well as non-tumor
cells).  If the relative levels of these cell types vary between the
"tumor" and "normal" tissue samples (and they always do), then of course
you will see gene expression differences.  Also, the very process of
removing a tumor (e.g. clamping off a vessel and therefore changing the
temperature and pH of the cells) will induce many gene expression
changes -- most of which are not really relevant to the oncogenic
"event".  Moreover, any tumor will be undergoing division -- so
expression of all the genes involved in cell replication and mitosis
(and there are lots) will be altered relative to the "normal" cells. Are
these cause or effect? In fact, even comparison of cloned cell lines
arrested in S or G1 phase of the cell cycle show numerous gene
expression changes.

I guess the point is that picking differntially expressed genes at
random from normal and tumor cells and trying to understand the
relevance of the genes to cancer is a dicey business at best.  Caveat
emptor!



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