cDNA preparation from <500 cells

[Michael Cooley]

Robert Hewitt (rhewitt at HELIX.NIH.GOV) wrote:
: Dear Internetter,

: We would like to get the Differential Display (Pardee amd Liang) on 
: microdissected samples of tissue which contain 500 or fewer cells.  

: So far we have the technique working on cell lines, where there is
: abundant starting material.  We have used a GITC-based protocol for total
: RNA preparation (Stratagene micro RNA preparation kit).  Our main concern
: with the microdissected material is making RNA from these small samples of
: tissue, and not loosing it all in the phenol extraction or isopropanol
: precipitation steps. We don't want to use tRNA as carrier, as it may give 
: bands in subsequent Differential Display.

: Has anyone had experience of making cDNA from fewer than 500 cells?  Any 
: tips would be much appreciated.

: Thank you in advance.

: Robert Hewitt


Hi, Eric Karr at CEPRAP, Univ. of Calif., Davis,CA 95616 (916-757-3045) 
was trying to make cDNA from 1 cell. Last I heard he was having some 
success, though it was difficult. 

My suggestion on precipitation is to use linear poly acrylamide instead 
of glycogen or tRNA. It doesn't interfer with the PCR


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