Guanidine-CsCl RNA microprep

[kim at m44.unm.edu]

In article <92192.170007FORSDYKE at QUCDN.QueensU.CA>, FORSDYKE at QUCDN.QueensU.CA writes:
>
>  For many years we have used successfully the method of Chirgwin and coworkers
>to prepare RNA from cultures of 10,000,000 to 100,000,000 lymphocytes
>(Forsdyke, 1984. Can.J.Biochem. 62, 859-864). RNA was separated from other
>macromolecules in a 5ml tube in a Beckman SW 50.1 rotor. Recently we tried to
>scale this down for 1,000,000 lymphocytes. We followed the procedure described
>by Brenner and workers (Biotechniques 7, 1096-1103). This employs the Beckman
>TL-100 bench-top ultracentrifuge and samples (0.2ml) are spun in an angle-head
>TLA-100 rotor for 2h at 80,000 r.p.m. Our recoveries of RNA were poor. We
>wonder if anyone out there has any information or references which might guide
>our troubleshooting of the method?
>                                  Sincerely,   Don Forsdyke, Queen's University
>                                                             Kingston, Canada.
Why not try the Acid Guanidinium Phenol Chloroform method of Chomzynski (sp)
and Sacci?  It is in Analytical Biochem. 162:156-159.  It  is easily scaled
down and does not require ultracentrifugation.  At the level of 10^6 cells, you
can probably scale it down to the level of an Eppendorf tube.

Daniel Kim