DD RT-PCR

Mary P. Remington mremingt at umabnet.ab.umd.edu
Wed Nov 26 07:53:50 EST 1997


     It has been my experience that the DD RT-PCR works better with cell
lines than tissue.  I base this on attempt to use both tissue and cell
lines.  I've tried several of the kits on the market and none of them are
a sure thing.  They generate a high number of false signals and
reproducibility is a problem as well.  I would recommend that if you have
a clue as to what you are looking for, I would get probes for those ideas
and do some RPAs or Northerns.  If you decide to proceed with DD RT-PCR I
would be sure to go with several rounds of subtraction.  
     I have tried the original protocol published in Science and found it
was not reproducible, but we did find a gene (Cytochrome C).  I tried the
Delta RNA Fingerprinting Kit w/rat brain and found it was not reproducible
and anchored primers didn't lend anything to the protocol.  I also tried
the PCR Select from Clontech and that did not work well with rat brain.  I
did find that RNAimage Kit from GenHunter Corp.  did have a degree of
reproducibility and I got different banding patterns using their anchored
primers (H-T11) and their arbitrary primers.  I sequenced some of the
fragments and several of the sequences were the same.  It seemed that the
smaller fragments in the gel were often times truncated amplifications of
larger fragments from the same lane but, the sequences were unique.  Note
that these findings were
with cells from a primary culture not tissue.  I have not yet tested these
fragments on a NOrthern or RPA to determine if they are real.  On a whole,
we have been disappointed with the protocol.  
     I attended the Differential Display meeting at Cold Spring Harbor
in Oct. '96 and most of the papers that found anything worth exploring
used cells not tissue.  Bottom line, you might be better off doing a
subtractive library.  Hope this input helps.  Mary  




More information about the Neur-sci mailing list