cDNA preparation from <500 cells

Donald W. Back dwback at
Tue Nov 29 18:56:33 EST 1994

In article <3bfo4u$h7a at> szcooley at (Michael Cooley) writes:
>From: szcooley at (Michael Cooley)
>Subject: Re: cDNA preparation from <500 cells
>Date: 29 Nov 1994 17:24:46 GMT

>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.  

>: 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

Try isolating the RNA using magnetic oligo(dT) beads, Dynal or polyATract from 
Promega, Microfast tract from InVitroGen or some other direct method.

As for making the cDNA you might want to look up an obscure reference.

Brady, G., Barbara, M. & Iscove, N.N. (1990) Representative in vitro cDNA 
amplification from individual hemopoietic cells and colonies. Meth. 
Molec.Cell.Biol. 2, 17-25.

Good luck,

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