primers in RT in situ PCR

[Shahram Mori]

Rob Wesley (rwesley at welchlink.welch.jhu.edu) wrote:
: Hi.

: I'm trying to work out a method for RT in situ PCR and I'm at a 
: conceptual snag.  In the RT step, I want to use the 'specific downstream 
: primer' but I'm not sure which one this is.  Is it the one complementary 
: to the 3' end of the mRNA?

YES. That's where the OligodT's bind. Poly A is added to the 3'end of mRNA's

  Can I use one of my PCR primers in this 
: step?  Would an oligo dT primer cause me all kinds of trouble?  How long 
: should the RT primer be?

Random hexamers and OligodT's are what you need. I think people over at John
Hopkins can help you. :)
: Robert Wesley
: Johns Hopkins University
: Department of Neurology
Cheers
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
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