RNA recovery from sorted cells
P.Openshaw Medicine (p.openshaw@ic.ac.uk)
Wed, 30 Nov 1994 12:15:02 +0000 (GMT)
We want to use RT-PCR to amplify mRNA from sorted cells, and are trying to
work out how to optimise for RNA recovery. We do not have a cooled
collection system on our Elite ESP, so to verify the purity of the
sorted cells (without capping off the stain) we are fixing after
staining, before the sort. Our hope is that the formalin will not affect
the RNA and that fixing the proteins will, if anything, inhibit RNA
destruction. An alternative might be to keep everything cold, but low
temperature apparently does not ensure RNA stability. We could collect
straight into cell lysis buffer with RNAase inhibitor, but could not then
verify recovery or purity. We are also considering sorting into tubes
containing vanadyl-ribonucleoside complexes or some other RNAase
inhibitor, with or without a permeablising agent, or fixing and stabilising
RNA prior to sorting.
Have others resolved this problem?
Peter Openshaw EMail: p.openshaw@ic.ac.uk
Respiratory Medicine Tel: +44 171 723 1252 x 5786
St. Mary's Med. School Fax: +44 171 724 7349
Imperial College of Science, Technology and Medicine
Norfolk Place, London W2 1PG, UK
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