cDNA preparation from <500 cells

Robert Hewitt rhewitt at HELIX.NIH.GOV
Sun Nov 27 08:50:03 EST 1994


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




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