[Q] mRNA display kits

Jon Nakamoto jnakamot at ucla.edu
Thu Sep 26 03:26:36 EST 1996


I can't believe I'm actually saying this, but the Clontech Delta RNA
Fingerprinting kit actually worked quite well for us (I've had mixed
results with some of their other products).

A med student working with me tried the Clontech kit for the first time
after spending 8 weeks with me learning differential display using a
homemade protocol and primers. Band consistency was quite good, although
we didn't get the large (> 1 kb) cDNAs their ads brag about (could be our
electrophoresis setup -- ask me in several weeks after I get my hands on a
genomyxLR sequencer). One thing I like is that the cDNA reaction is done
with a single oligo-dT primer; the different combinations of arbitrary and
selective anchored oligo-dT primers are used during the PCR part of the
reaction. We did follow their protocol to the letter, including using
their rather expensive Klen-Taq/Taq Start polymerase mix. No question that
the longer primers do work better (the ability to increase your annealing
temperature after the first few cycles at 40 degrees undoubtedly makes a
big difference in reproducibility). 

I have in hand, but haven't yet tried the Stratagene RAP-PCR kit (I was
interested in running some side-by-side comparisons, since I'm getting a
bit tired of so many 3' UTR sequences, and thought that the RAP-PCR
approach might help, even if there's a bit more variability from using
dual arbitrary primers). Anybody out there have hands-on experience with
RAP-PCR vs. oligo-dT anchored differential display comparisons?

I'm sure that GenHunter, Display Systems, Genomyx, and who knows how many
others are constantly upgrading their kits to include the latest and
greatest things (longer primers, antisense strands with T7 RNA pol
promoters for use to make probes/RNase protection, convenient sequencing
primer sites, etc). What they all have in common so far is high price, and
I recommend finding someone in Montreal who will let you spend a couple of
days watching and learning his/her successful differential display
protocol in action, after which you can spend your precious money buying
reasonable quantities of enzyme, nucleotides, and primers, rather than the
micro-amounts generally provided in most kits. A homemade protocol also
tends to be a bit more flexible in terms of introducing the latest
technical improvements -- I know I'll be trying silver staining soon a la
the most recent Biotechniques article, so that I can hopefully save on 33P
and cut out bands more directly (and therefore accurately). 

But a kit can get you started quickly, no doubt. For those of us with only
a little money but even less time, this has great appeal (as long as you
don't get lazy and become completely dependent, buying expensive kit after
kit rather than setting up your own more economical protocol). 

I have no connection with Clontech or any other of the above-mentioned
companies, and views are my own, etc, etc.

Jon

Jon Nakamoto, MD
Asst Clin Prof of Pediatrics/Endocrinology
UCLA Children's Hospital

jnakamot at ucla.edu



In article <52bsr3$le0 at epervier.CC.UMontreal.CA>, pions at ERE.UMontreal.CA
(Pion Stephane) wrote:

> This is a simple question actually.  Has anyone used any good mRNA
display kits
> that are on the market?  If so, which ones.  I'm looking for a reliable kit
> and so i thought i"d ask around.
> 
> Thanks for the information
> 
> Stephane Pion
> pions at ere.umontreal.ca

-- 
Jon Nakamoto
jnakamot at ucla.edu



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