RT-PCR of Blood Samples

[Dom Spinella]

Eduardo Wainstein wrote:
> We are planning to screen patients for the expression of T receptor 
> isoforms in linfocytes using RT-PCR. Could anybody give me an idea 
> of how much blood should I require per reaction? We plan to perform up to 40 reactions per patient.

Eduardo:

If by "T-cell receptor isoforms" you mean performing a repertoire 
analysis of V-alpha or V-beta gene expression, this is not a trivial 
PCR application.  You need to start with at least 1 ml of blood -- and 
preferably 10 ml or more to prevent sampling artifacts in the use of 
individual V-elements.  There are also a number of other 
considerations that you need to be aware of in order get an accurate 
assessment of T-cell repertories.  You might check out a book chapter 
I wrote on the subject (as well as references cited therein) called 
"Analysis of T-cell Repertoires by PCR" by Dominic G. Spinella and 
James M. Robertson in "The Polymerase Chain Reaction", Kary Mullis, 
Francois Ferre, and Richard Gibbs, eds.  Birkhauser, Boston MA, 1994 
pp. 110-120.  If you e-mail me an address, I'll send you a reprint.  
Good luck.

D.G. Spinella, Ph.D.