cDNA preparation from <500 cells
Donald W. Back
dwback at hookup.net
Tue Nov 29 18:56:33 EST 1994
In article <3bfo4u$h7a at mark.ucdavis.edu> szcooley at chip.ucdavis.edu (Michael Cooley) writes:
>From: szcooley at chip.ucdavis.edu (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.
More information about the Methods