Help with DD RT-PCR??

[Louis Alarcon]

In article <DvJ5LF.BpD at midway.uchicago.edu>, Alex Haddad
<mahaddad at midway.uchicago.edu> wrote:

> ...I am mostly concerned with the PCR steps, i.e., good primer 
> combinations, appropriate controls, ect. and the post-band 
> identification analysis, such as what is the most efficient and 
> high-throughput way to clone DD products, confirm their induction 
> patterns, ect...
> 
> Alex Haddad

Some answers:

1) We do the DD-PCR step as specified by Mou et al in BBRC 199(2):564, 1994.
   and use 33P dATP (instead of 35S) -> better band resolution.
2) The optimal 5' decamer primers have 50% GC content.  There was one
report        
   that found the best primers had a G or C as the first base, while the least
   effective ended in G or C (I can't remeber that reference now...).  Genosys
   has several different kits of decamers designed specifically for DD-PCR.
3) The optimal negative control depends on the stimulated condition you wish to 
   study... you did not mention this.
4) We clone our excised, eluted, re-amplified bands in a TA cloning vector from
   Invitrogen (Promega and others also have them).  It has been very simple and 
   effective.
5) The confirmation of differential expression can be done with Northern blot. 
   Recognizing its limited sensitivity, if northern fails to show any 
   expression, ribonuclease protection assay or hot PCR with gene-specific 
   primers can overcome that problem.
6) Bands of interest are sequenced in the plasmid, and used to scan Genbank 
   through the NCBI website.  Isolation of the entire transcript can be done by
   screening an expression library, or, perhaps faster, by 5' RACE.
7) Good luck and happy gene hunting!!!

lou alarcon
univ of pittsburgh dept of surgery