RNA Differential Display
T. S. Grewal
sasgrewl at reading.ac.uk
Thu Aug 24 11:11:27 EST 1995
There are a lot of DDRT-PCR kits out at the moment. The one we opted
for was the 'Delta RNA Fingerprinting' kit from ClonTech. It's main
advantages over the other kits is that:
1) You carry out only 1 cDNA synthesis per RNA sample
2) It is highly reproducible (a very important point)
3) You only need to use 20-25 cycles (normally 40 cycles are used in other
kits) this means you get less non-specific products or mis-priming.
4) The primer combinations (2 x 10 primers) are more than provided
in some kits.
This system has worked where other ones we tried did not give good results.
The other points to keep in mind when doing DDRT-PCR:
1) You need very pure DNA-free RNA, we use a reagent that gives very
good quality RNA that's OK to use in DDRT-PCR and takes 1 hr to
process 1-20 tissue samples.
2) Use a good quality Taq enzyme - we got the best results with
the Stoffel fragment of AmpliTaq.
3) Use some form of HotStart PCR (eg WaxBeads or TaqStart Antibody)
4) Use 33-P dATP as the label.
The whole process from tissue to RNA, to cDNA and PCR to results on a
polyacrylamide gel autoradiograph takes 2.5 days. Even if you have
never done this type of work before.
Hope this has been of some help. Good luck.
Dr. T.S. Grewal
University of Reading.
On Fri, 18 Aug 1995, Robyn A. Kerr wrote:
> Is there anyone out there doing RNA Differentail Display? We are considering
> settinng up the technique but do not have any experience and need all the
> advice we can get!
More information about the Methods